A method for detecting ningnanmycin by high performance liquid chromatography

Abstract

This invention relates to a high-performance liquid chromatography (HPLC) method for detecting ningnanmycin. By using a specific chromatographic column and mobile phase, excellent resolution, accuracy, and precision are achieved. The detection method of this invention enables excellent separation of ningnanmycin from structurally similar homologues, greatly improving the accuracy and efficiency of ningnanmycin detection.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection and analysis technology, specifically relating to a method for detecting ningnanmycin by high performance liquid chromatography. Background Technology

[0002] Ningnanmycin was isolated from soil in Ningnan County, Sichuan Province. It is a newly discovered cytosine nucleoside peptide antibiotic, hence the name Ningnanmycin for its fermentation product. A series of Ningnanmycin products have been developed, including 2% aqueous solution, 8% aqueous solution, 10% soluble powder, and a bitter-Ning seed dressing agent. It has been registered for use against tobacco mosaic virus, tomato virus, pepper virus, rice damping-off, soybean root rot, rice stripe leaf blight, apple leaf spot, and cucumber powdery mildew. Currently, there are 11 registration certificates in China, including two for 40% Ningnanmycin technical material. In addition, Ningnanmycin has been widely used to control rapeseed sclerotinia stem rot, litchi downy mildew, and various other crop diseases such as viral diseases, stem rot, vine blight, and powdery mildew.

[0003] Ningnanmycin is an antibiotic pesticide produced using microbial fermentation technology. Its characteristic feature is that its producing strain, 16A-6, is a new variant of *Streptomyces noursei*, named *Streptomyces noursei var. xichangensisn var.*. The antibiotic produced by this bacterium is named Ningnanmycin, with the chemical name 1-(4-sarcosamide-L-seramide-4-deoxy-β-D-glucuronide)cytosine, a molecular weight of 444 [mass spectrometry], and the molecular formula C1. 16 H 25 N7O8. The structural formula is:

[0004]

[0005] Based on currently available information, most enterprise standards adopt high-performance liquid chromatography (HPLC). However, according to our actual testing, the feasibility of these publicly available methods is not high. Firstly, there is a lack of supporting standards; secondly, the spectral data of some methods do not match the actual results, indicating low reproducibility. Having purified and prepared ningnanmycin standards in our laboratory, we studied a high-performance liquid chromatography method for the detection of ningnanmycin. The linear relationship of this ningnanmycin detection method was verified as y = 2850.7x - 9297.2, with a linear coefficient R0. 2 =0.9998; the accuracy test recovery rate ranged from 99.1% to 100.8%, with an average recovery rate of 99.8%; the precision test RSD was 0.58%, which was less than the calculated value of 2.07 by Horwitz equation 2(1-0.5logC)×0.67; the detection limit was approximately 6 ppm, and all validation parameters met the requirements for the detection of ningnanmycin samples. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for detecting ningnanmycin using high-performance liquid chromatography (HPLC). This method enables the separation of ningnanmycin, thereby achieving product quality control.

[0007] To achieve the purpose of this invention, the following technical solution is adopted: a method for detecting ningnanmycin by high performance liquid chromatography, using the following chromatographic conditions:

[0008] Column: The stationary phase is silica gel bonded with alkylsilanes containing embedded polar groups;

[0009] Mobile phase: Ammonium acetate + sodium dihydrogen phosphate aqueous solution, pH adjusted with phosphoric acid;

[0010] Flow rate: 0.4-1.0 mL / min;

[0011] Detection wavelength: 266-268nm;

[0012] Column temperature: 10-40℃;

[0013] Injection volume: 5-20 μL;

[0014] Detector: Variable light ultraviolet detector or diode array detector.

[0015] In a preferred embodiment of the present invention, a chromatographic column preferably uses octadecylsilane-bonded silica gel with embedded polar groups as the stationary phase.

[0016] In a preferred embodiment of the present invention, the mobile phase contains ammonium acetate, and the molar concentration of ammonium acetate is in the range of 0.003 mol / L to 0.010 mol / L, preferably 0.005 mol / L.

[0017] In a preferred embodiment of the present invention, the mobile phase further contains sodium dihydrogen phosphate, wherein the mass concentration of sodium dihydrogen phosphate ranges from 0.1% to 0.3%, preferably 0.2%.

[0018] In a preferred embodiment of the present invention, the mobile phase is further adjusted to pH with phosphoric acid, with a pH range of 4.5 to 6.0, preferably 5.5.

[0019] In a preferred embodiment of the present invention, the flow rate is 0.5 mL / min; the detection wavelength is 267 nm; the column temperature is 30 °C; and the injection volume is 10 μL.

[0020] In a preferred embodiment of the present invention, the chromatographic column and the mobile phase are used in combination.

[0021] Furthermore, the method is implemented through the following steps:

[0022] (1) Weigh out an appropriate amount of ningnanmycin standard, dissolve it in pure water, make up to volume, shake well, filter through a 0.45μm filter membrane, and prepare the test standard solution;

[0023] (2) Weigh a sample containing an appropriate amount of ningnanmycin, dissolve it in pure water, make up to volume, shake well, filter it through a 0.45μm filter membrane, and prepare the test sample solution;

[0024] (3) Take the test standard and sample solution, inject them into the high performance liquid chromatograph, and perform high performance liquid chromatography analysis according to the chromatographic conditions described.

[0025] Compared with existing technologies, this invention achieves excellent resolution, accuracy, and precision through the combined use of the specific chromatographic column and mobile phase described above; the detection method of this invention enables excellent separation of ningnanmycin from structurally similar homologues, greatly improving the accuracy and efficiency of ningnanmycin detection; using the detection method of this invention, the peak elution time of ningnanmycin is approximately 4.5-5.3 min. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings:

[0027] Figure 1 The image shows a high-performance liquid chromatogram of a ningnanmycin sample separated according to the conditions of Example 1.

[0028] Figure 2 The image shows a high-performance liquid chromatogram of a ningnanmycin sample separated according to the conditions of Example 2.

[0029] Figure 3 The image shows a high-performance liquid chromatogram of a ningnanmycin sample separated according to the conditions of Example 3.

[0030] Figure 4 This is a linear relationship diagram of the analysis method of the present invention. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0032] Example 1

[0033] Experimental instruments and conditions:

[0034] Shimadzu 20A high-performance liquid chromatograph with diode array detector;

[0035] Chromatographic column: RPC18 (250×4.6mm, 5μm);

[0036] Mobile phase: (0.005 mol / L ammonium acetate + 0.2% sodium dihydrogen phosphate) aqueous solution, adjusted to pH 5.5 with phosphoric acid.

[0037] Flow rate: 0.5 mL / min;

[0038] Detection wavelength: 267nm;

[0039] Column temperature: 30℃;

[0040] Injection volume: 10 μL.

[0041] Experimental steps:

[0042] Weigh out 0.02g of ningnanmycin standard into a 50mL volumetric flask, dissolve and dilute to volume with pure water, shake well, filter through a 0.45μm filter membrane, and prepare the test standard solution;

[0043] Weigh a sample containing 0.02g of ningnanmycin into a 50mL volumetric flask, dissolve and dilute to volume with pure water, shake well, filter through a 0.45μm filter membrane, and prepare the test sample solution;

[0044] Take the above-mentioned test standard and sample solution, inject them into the high-performance liquid chromatograph (HPLC), and perform HPLC analysis according to the above chromatographic conditions. Record the chromatograms. Results are shown below. Figure 1 ,like Figure 1 As shown, the elution time of ningnanmycin was 5.3 min.

[0045] Example 2

[0046] Experimental instruments and conditions:

[0047] Shimadzu 20A high-performance liquid chromatograph with diode array detector;

[0048] Chromatographic column: RPC18 (250×4.6mm, 5μm);

[0049] Mobile phase: (0.005 mol / L ammonium acetate + 0.2% sodium dihydrogen phosphate) aqueous solution, adjusted to pH 5.3 with phosphoric acid.

[0050] Flow rate: 0.5 mL / min;

[0051] Detection wavelength: 267nm;

[0052] Column temperature: 30℃;

[0053] Injection volume: 10 μL.

[0054] Experimental steps:

[0055] Weigh out 0.02g of ningnanmycin standard into a 50mL volumetric flask, dissolve and dilute to volume with pure water, shake well, filter through a 0.45μm filter membrane, and prepare the test standard solution;

[0056] Weigh a sample containing 0.02g of ningnanmycin into a 50mL volumetric flask, dissolve and dilute to volume with pure water, shake well, filter through a 0.45μm filter membrane, and prepare the test sample solution;

[0057] Take the above-mentioned test standard and sample solution, inject them into the high-performance liquid chromatograph (HPLC), and perform HPLC analysis according to the above chromatographic conditions. Record the chromatograms. Results are shown below. Figure 2 ,like Figure 2 As shown, the elution time of ningnanmycin was 4.7 min.

[0058] Example 3

[0059] Experimental instruments and conditions:

[0060] Shimadzu 20A high-performance liquid chromatograph with diode array detector;

[0061] Chromatographic column: RPC18 (250×4.6mm, 5μm);

[0062] Mobile phase: (0.005 mol / L ammonium acetate + 0.25% sodium dihydrogen phosphate) aqueous solution, adjusted to pH 5.5 with phosphoric acid.

[0063] Flow rate: 0.5 mL / min;

[0064] Detection wavelength: 268nm;

[0065] Column temperature: 30℃;

[0066] Injection volume: 10 μL.

[0067] Experimental steps:

[0068] Weigh out 0.02g of ningnanmycin standard into a 50mL volumetric flask, dissolve and dilute to volume with pure water, shake well, filter through a 0.45μm filter membrane, and prepare the test standard solution;

[0069] Weigh a sample containing 0.02g of ningnanmycin into a 50mL volumetric flask, dissolve and dilute to volume with pure water, shake well, filter through a 0.45μm filter membrane, and prepare the test sample solution;

[0070] Take the above-mentioned test standard and sample solution, inject them into the high-performance liquid chromatograph (HPLC), and perform HPLC analysis according to the above chromatographic conditions. Record the chromatograms. Results are shown below. Figure 3 ,like Figure 3 As shown, the elution time of ningnanmycin was 4.5 min.

[0071] Linear correlation experiment of analytical methods

[0072] Weigh approximately 0.1 g of ningnanmycin standard into a 50 ml volumetric flask, dissolve and dilute to volume with ultrapure water. Transfer 2 ml, 5 ml, 10 ml, 15 ml, and 20 ml of this solution into five separate 50 ml volumetric flasks, dissolve and dilute to volume, preparing the ningnanmycin standard solutions as shown in the table below. After the instrument has stabilized, inject the solutions in ascending order of concentration. Construct a standard curve with ningnanmycin concentration on the x-axis and peak area on the y-axis.

[0073] As shown in Table 1 below, the measured standard curve is: Y = 2850.7X - 9297.2, and the linear correlation coefficient is R. 2 =0.9998. This demonstrates that ningnanmycin exhibits good linearity within the concentration range of 95.266 μg / mL to 952.662 μg / mL. The linearity graph is shown below. Figure 4 As shown.

[0074] Table 1. Experimental results of linear correlation analysis method

[0075]

[0076]

[0077] Accuracy test of analytical methods

[0078] Five samples were weighed from a ningnanmycin sample with a known mass fraction, and a certain amount of ningnanmycin standard was added to each sample. The samples were analyzed according to the detection method of this invention. As shown in Table 2, the recovery rate of ningnanmycin was found to be in the range of 99.1% to 100.8%, with an average recovery rate of 99.8%.

[0079] Table 2. Accuracy test results of the analytical methods

[0080]

[0081] Precision test of analytical method

[0082] Five samples were accurately weighed from the same sample and analyzed according to the detection method of this invention. As shown in Table 3, the relative standard deviation of ningnanmycin after five parallel determinations under this detection method was 0.58%, which is less than the calculated value of 2.07 by Horwitz equation 2(1-0.5logC)×0.67, proving that the precision of this method meets the requirements for method validation.

[0083] Table 3. Precision test results of the analytical methods

[0084]

[0085] Sensitivity test of analytical method

[0086] Sample A1 was serially diluted (2x, 4x, 8x, 16x, etc.) and tested one by one until the active ingredient was undetectable. As shown in Table 4, the data indicates that the limit of detection for ningnanmycin is approximately 6 ppm, which is sufficient for general routine detection and is suitable for the detection of ningnanmycin technical and related formulations.

[0087] Table 4. Sensitivity test results of the analytical methods

[0088]

[0089] The above experimental results show that the detection method of the present invention has high accuracy and precision, good linearity, and a wide range of applicable sensitivity.

[0090] Furthermore, compared with existing liquid chromatography conditions, the liquid chromatography conditions of the present invention have better separation and reproducibility for ningnanmycin, thereby ensuring the accuracy of detection.

[0091] The above preferred embodiments are only used to illustrate the content of this invention patent. In addition, there are other embodiments of this invention. Any technical solutions formed by those skilled in the art through equivalent substitution or equivalent modification based on the technical teachings involved in this invention fall within the protection scope of this invention.

Claims

1. A method for detecting ningnanmycin by high performance liquid chromatography, characterized in that, The following chromatographic conditions were used: Column: The stationary phase is octadecylsilane-bonded silica gel with embedded polar groups; Mobile phase: Ammonium acetate + sodium dihydrogen phosphate aqueous solution, pH adjusted with phosphoric acid, pH range 4.5~6.0; Flow rate: 0.4-1.0 mL / min; Detection wavelength: 266-268nm; Column temperature: 10-40℃; Injection volume: 5-20µL; Detector: Variable light ultraviolet detector or diode array detector; The mobile phase contains ammonium acetate, with a molar concentration ranging from 0.003 mol / L to 0.010 mol / L; the mobile phase also contains sodium dihydrogen phosphate, with a mass concentration ranging from 0.1% to 0.3%.

2. The method according to claim 1, characterized in that, The molar concentration of ammonium acetate is 0.005 mol / L.

3. The method according to claim 1, characterized in that, The mass concentration of sodium dihydrogen phosphate is 0.2%.

4. The method according to claim 1, characterized in that, The mobile phase also needs to be adjusted to pH 5.5 with phosphoric acid.

5. The method according to claim 1, characterized in that, The flow rate was 0.5 mL / min; the detection wavelength was 267 nm; the column temperature was 30 °C; and the injection volume was 10 µL.

6. The method according to any one of claims 1-5, characterized in that, The chromatographic column and mobile phase are used together.

7. The method according to claim 6, characterized in that, The method is implemented through the following steps: (1) Weigh out an appropriate amount of ningnanmycin standard, dissolve it in pure water, make up to volume, shake well, filter through a 0.45µm filter membrane, and prepare the test standard solution; (2) Weigh a sample containing an appropriate amount of ningnanmycin, dissolve it in pure water, make up to volume, shake well, filter through a 0.45µm filter membrane, and prepare the test sample solution; (3) Take the test standard and sample solution, inject them into the high performance liquid chromatograph, and perform high performance liquid chromatography analysis according to the chromatographic conditions described.

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