CN-116223665-B - Method for detecting Ningnanmycin by high performance liquid chromatography

Abstract

The invention relates to a method for detecting Ningnanmycin by high performance liquid chromatography, which obtains good separation degree, accuracy and precision through the matched use of a specific chromatographic column and a mobile phase; by adopting the detection method, the ningnanmycin and the homologs with similar structures can be well separated, and the accuracy and the detection efficiency of the detection of the ningnanmycin are greatly improved.

Inventors

• YANG HONGBO
• ZHANG NAN
• LI HAOYU
• MA WENYAN
• ZHENG PENGFEI
• LI SHUANGSHUANG
• PAN ZHONGCHENG

Assignees

• 陕西麦可罗生物科技有限公司

Dates

Publication Date

20260505

Application Date

20230207

Claims (7)

1. 1. A method for detecting Ningnanmycin by high performance liquid chromatography is characterized in that the following chromatographic conditions are adopted: a chromatographic column takes octadecylsilane chemically bonded silica gel with embedded polar groups as a stationary phase; the mobile phase is ammonium acetate and sodium dihydrogen phosphate aqueous solution, and the pH value is regulated by phosphoric acid, wherein the pH value range is 4.5-6.0; The flow rate is 0.4-1.0mL/min; The detection wavelength is 266-268nm; column temperature is 10-40 ℃; the sample injection amount is 5-20 mu L; a detector, a variable light ultraviolet detector or a diode array detector; The mobile phase contains ammonium acetate, the molar concentration range of the ammonium acetate is 0.003 mol/L-0.010 mol/L, and the mobile phase also contains sodium dihydrogen phosphate, and the mass concentration range of the sodium dihydrogen phosphate is 0.1-0.3%.
2. 2. The method according to claim 1, wherein the molar concentration of ammonium acetate is 0.005mol/L.
3. 3. The method according to claim 1, wherein the mass concentration of sodium dihydrogen phosphate is 0.2%.
4. 4. The method of claim 1, wherein the mobile phase is further pH adjusted with phosphoric acid to a pH of 5.5.
5. 5. The method according to claim 1, wherein the flow rate is 0.5mL/min, the detection wavelength is 267nm, the column temperature is 30 ℃, and the sample introduction amount is 10. Mu.L.
6. 6. The method of any one of claims 1-5, wherein the chromatography column and the flow are used in combination.
7. 7. The method according to claim 6, wherein the method is implemented by: (1) Weighing a standard substance containing a proper amount of ningnanmycin, dissolving with pure water, fixing the volume, shaking uniformly, and filtering by a 0.45 mu m filter membrane to prepare a test standard substance solution; (2) Weighing a sample containing a proper amount of Ningnanmycin, dissolving with pure water, fixing the volume, shaking uniformly, and filtering through a 0.45 mu m filter membrane to prepare a sample solution; (3) And (3) taking a sample to be tested and a sample solution, injecting the sample solution into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions.

Description

Method for detecting Ningnanmycin by high performance liquid chromatography Technical Field The invention belongs to the technical field of pesticide detection and analysis, and particularly relates to a method for detecting ningnanmycin by using a high performance liquid chromatography. Background Ningnan mycin is obtained by separating soil in Ningnan county of Sichuan province and is a novel cytosine nucleoside peptide antibiotic discovered for the first time, so the fermentation product is named as Ningnan mycin. Ningnan mycin series products such as 2% aqueous solution, 8% aqueous solution, 10% soluble powder and bitter-Ning Chongyi agent have been developed, and are registered on tobacco mosaic virus diseases, tomato virus diseases, pepper virus diseases, rice damping-off diseases, soybean root rot diseases, rice stripe leaf blight diseases, apple alternaria leaf spot and cucumber powdery mildew, and the total number of domestic registration certificates is 11, wherein the number of the registration certificates of 40% Ningnan mycin is two. In addition, ningnan mycin is widely popularized and applied in preventing and treating sclerotinia rot of colza, downy mildew of litchi, virus diseases, stem rot, gummy stem rot, powdery mildew and other diseases of other crops. Ningnanmycin is an antibiotic pesticide produced by microbial fermentation technology, and is characterized in that the producing strain 16A-6 is a new variety of Streptomyces nordsi, named Streptomyces nordsi West variety (Streptomycesnourseivar xichangensisn var). The antibiotic produced by the bacterium is named Ningnanmycin, the chemical name of which is 1- (4-myoamino-L-serinamide-4-deoxy-beta-D-glucopyranose aldehyde amide) cytosine, the molecular weight of which is 444[ mass spectrometry ], and the molecular formula of which is C 16H25N7O8. The structural formula is as follows: From the presently disclosed data, most enterprise standards adopt liquid chromatography, but according to our practical detection, the feasibility of the disclosed detection method is not high, firstly, the standard product is lack of support for the detection method, secondly, the relevant spectrogram of part of the detection method is not consistent with the practical detection, and the reproducibility is not high. On the premise of purifying and preparing the Ningnanmycin standard product in the laboratory, the high-efficiency liquid phase detection method of Ningnanmycin is researched. The linear relation of the Ningnanmycin detection method is verified to be y= 2850.7x-9297.2, the linear coefficient is R 2 =0.9998, the accuracy test recovery rate is in the range of 99.1% -100.8%, the average recovery rate is 99.8%, the accuracy test RSD is 0.58% and is smaller than the calculated value of Horwitz equation 2 (1-0.5 log C) multiplied by 0.67 and 2.07, the detection limit is about 6ppm, and all verification parameters meet the detection of Ningnanmycin samples. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a method for detecting Ningnanmycin by using a high performance liquid chromatography, and the method can realize separation of Ningnanmycin, thereby realizing control of product quality. In order to achieve the aim of the invention, the invention adopts the following technical scheme, and the method for detecting the ningnanmycin by using the high performance liquid chromatography adopts the following chromatographic conditions: Chromatographic column with embedded polar group alkyl silane bonded silica gel as stationary phase; Mobile phase, ammonium acetate and sodium dihydrogen phosphate aqueous solution, and regulating pH with phosphoric acid; The flow rate is 0.4-1.0mL/min; The detection wavelength is 266-268nm; column temperature is 10-40 ℃; the sample injection amount is 5-20 mu L; detector, variable light ultraviolet detector or diode array detector. In a preferred embodiment of the present invention, a column with octadecylsilane chemically bonded silica having an embedded polar group as a stationary phase is preferred. In a preferred embodiment of the invention, the mobile phase contains ammonium acetate in a molar concentration range of 0.003mol/L to 0.010mol/L, preferably 0.005mol/L. In a preferred embodiment of the invention, the mobile phase also contains sodium dihydrogen phosphate, the mass concentration of which ranges from 0.1% to 0.3%, preferably 0.2%. In a preferred embodiment of the present invention, the mobile phase is further adjusted to a pH in the range of 4.5 to 6.0, preferably 5.5, with phosphoric acid. In a preferred embodiment of the invention, the flow rate is 0.5mL/min, the detection wavelength is 267nm, the column temperature is 30 ℃ and the sample injection amount is 10 mu L. In a preferred embodiment of the invention, the chromatography column and the mobile phase are used in combination. Further, the method is realized by the following steps: (1) Weighing a standard sub