Separation method of nystatin and components thereof

Abstract

The invention discloses a separation method of nystatin and components thereof. The method comprises the step of determining nystatin and components thereof in a test sample by using a high performance liquid chromatograph. The separation and detection method disclosed by the invention is good in separation degree, excellent in peak pattern and simple to operate, and also has good method specificity and sensitivity.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a separation and detection method for nystatin components. Background Art

[0002] Nystatin, whose molecular formula is C 47 H 75 NO 17 , molecular weight is 926.095.

[0003] Nystatin is a polyene antibiotic with a conjugated polyene macrolide structure. It has a broad-spectrum antifungal effect and can inhibit the activity of fungi and dermatophytes, but has no inhibitory effect on bacteria.

[0004] It is mainly used for internal treatment of digestive tract fungal infections or external use for surface skin fungal infections, such as fungal gastritis in cattle, fungal disease in chickens and turkeys, etc. It is also effective for mastitis caused by Aspergillus and Mucor through milk duct perfusion. It is also effective for pneumonia in chicks caused by Aspergillus fumigatus through spray inhalation. This product is also used for fungal superinfection caused by long-term use of broad-spectrum antibiotics. Summary of the invention

[0005] In view of the above problems existing in the prior art, in order to ensure that the quality of nystatin can be effectively controlled, The purpose of the present invention is to provide a method for separating and detecting nystatin and its related components, which has high sensitivity and strong specificity.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a method for separating nystatin and its components, the method comprising using a high performance liquid chromatograph to determine the nystatin components in a test sample.

[0007] Preferably, the mobile phase A of the HPLC is acetonitrile-0.05M ammonium acetate (29:71), and the mobile phase B is acetonitrile-0.05M ammonium acetate (60:40).

[0008] Preferably, the volume ratio of acetonitrile to 0.05 M ammonium acetate in mobile phase A is 29:71, and the volume ratio of acetonitrile to 0.05 M ammonium acetate in mobile phase B is 60:40.

[0009] Preferably, the detection wavelength of the high performance liquid chromatograph is 305 nm.

[0010] Preferably, the elution conditions of the high performance liquid chromatograph are: Time (min) A% B% 0 95 5 30 95 5 40 60 40 50 60 40 51 55 5 60 95 5 Preferably, the column temperature of the high performance liquid chromatograph is 30°C.

[0011] Preferably, the injection volume of the high performance liquid chromatograph is 20 μl.

[0012] Preferably, the chromatographic column of the high performance liquid chromatography is Welch Ultimate XB-C18, 4.6×150 mm, 5 μm.

[0013] Effects of the Invention The invention provides a separation and detection method for nystatin and its components, which has good separation degree, excellent peak shape, simple operation, and good method specificity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 HPLC chromatogram of the specificity experiment of Example 1 of the present invention DETAILED DESCRIPTION

[0015] The following examples are only used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention.

[0016] Example Inspection method: Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia) (1) Reagent solutions and reference substances Methanol (HPLC grade) Acetonitrile (HPLC grade) Dimethyl sulfoxide (HPLC grade) Ammonium Acetate (AR Grade) Water (purified water) (2) Instruments and equipment High performance liquid chromatography Balance (3) Preparation of test solution and control solution Solvent: dimethyl sulfoxide.

[0017] Blank auxiliary material: Take about 200 mg of blank auxiliary material, accurately weigh it, put it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0018] Test solution: Take about 200 mg of the product, accurately weigh it, put it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0019] (4) System suitability solution Take about 20 mg of the raw material, weigh it accurately, put it in a 50 ml volumetric flask, add 25 ml of methanol to dissolve it, mix it well, add water to dilute it to the scale, and shake it well; take 10 ml of the solution, add 2.0 ml of dilute hydrochloric acid, mix it well, and let it stand at room temperature for 1 hour.

[0020] (5) Sensitivity solution Accurately measure 1 ml of the control solution, place it in a 20 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0021] Accurately measure the blank solution (i.e. solvent), blank excipient, sensitivity solution, system suitability solution, and test solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0022] Example 1 - Specificity Experiment Solvent: dimethyl sulfoxide.

[0023] Blank auxiliary material: Take about 200 mg of blank auxiliary material, accurately weigh it, put it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0024] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 20 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0025] System suitability solution: Take about 20 mg of the raw material, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of methanol to dissolve, mix well, dilute to the scale with water, and shake well; take 10 ml of the solution, add 2.0 ml of dilute hydrochloric acid, mix well, and let stand at room temperature for 1 hour.

[0026] Test solution: Take about 200 mg of the product, accurately weigh it, put it in a 25 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0027] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0028] Take blank solvent, blank auxiliary material solution, sensitivity solution, system suitability solution, and test solution and measure them according to the law, record the chromatogram, Figure 1 HPLC chromatogram of the specificity experiment of Example 1 of the present invention.

[0029] Specificity test results The blank solution, blank excipients and each component solution had no interference on the detection of nystatin; in the sensitivity solution chromatogram, the signal-to-noise ratio of the nystatin peak was 20, which was greater than 10; in the system suitability solution chromatogram, the separation degree of the two main peaks was 31, which was greater than 3.5.

Claims

1. A method for separating nystatin and its components, characterized in that: The method comprises the step of using a high performance liquid chromatograph to determine the nystatin component in the test sample.

2. The separation method according to claim 1, characterized in that The mobile phase A of the HPLC is acetonitrile-0.05M ammonium acetate (29:71), and the mobile phase B is acetonitrile-0.05M ammonium acetate (60:40).

3. The separation method according to claim 1, wherein the volume ratio of acetonitrile to 0.05 M ammonium acetate in mobile phase A is 29:71, and the volume ratio of acetonitrile to 0.05 M ammonium acetate in mobile phase B is 60:

40.

4. The separation method according to claim 1, characterized in that The detection wavelength of the high performance liquid chromatograph is 305 nm.

5. The separation method according to claim 1, characterized in that The elution conditions of the high performance liquid chromatograph are:

6. The separation method according to claim 1, characterized in that The column temperature of the high performance liquid chromatograph is 20-40°C.

7. The separation method according to claim 1, characterized in that The injection volume of the high performance liquid chromatograph is 5-20 μl.

8. The separation method according to claim 1, characterized in that The chromatographic column of the high performance liquid chromatography is Welch Ultimate XB-C18, 4.6×150 mm, 5 μm.

9. The separation method according to claim 1, characterized in that The flow rate of the high performance liquid chromatograph is 1.0 ml / min.

Images