Rapid qualitative identification method for sildenafil compounds in food based on nuclear magnetic resonance technology

Abstract

The invention provides a rapid qualitative identification method for sildenafil compounds in food based on a nuclear magnetic resonance technology, which comprises the following steps: collecting a sildenafil compound standard sample map, processing the sildenafil compound standard sample map by topspin / CMC-assist software, importing the sildenafil compound standard sample map into AssureNMR, and establishing a sildenafil substance standard nuclear magnetic spectrum library; and importing a sildenafil to-be-tested sample map to be matched with a database result in a simulation manner. After a part of sildenafil substances are selected to build a library, a standard sample of a certain specific substance is not needed in subsequent detection, the expenditure for purchasing a large number of standard samples and the waiting period are saved, rapid detection and identification can be achieved under the condition that no standard substance exists, and the situation that lawbreakers modify compound groups to escape supervision is powerfully deterred.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a method for rapid qualitative identification of sildenafil compounds in food based on nuclear magnetic resonance technology. Background Art

[0002] Food safety is a serious challenge facing society today. Adding harmful substances to food for profit is a common occurrence, and ensuring the safety of residents is urgent. Sildenafil-like substances are non-food ingredients that are expressly prohibited by the state from being added to food and health supplements. However, current market research indicates that many unscrupulous businesses still add sildenafil-like substances to food or food ingredients. Rapid detection of their presence is crucial for ensuring food safety.

[0003] Currently, conventional detection methods include HPLC / LC-MS / MS methods, which usually require the preparation of a large number of solvents or eluents during the experimental stage, including high-risk reagents such as hydrochloric acid and formic acid, posing potential risks to the health and safety of experimental personnel. Moreover, existing technologies usually require standard samples of target compounds as controls to establish qualitative and quantitative methods, which hinders the qualitative identification of unknown compounds in real cases. In addition, the HSQC method has a long sampling time, complicated experimental parameters, and requires a specific pulse sequence, which increases the time and labor costs of detection. Moreover, the results are not subjected to mathematical and statistical analysis, which may increase the risk of misjudgment. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the prior art to a certain extent. To this end, one object of the present invention is to provide a method for rapid qualitative identification of sildenafil compounds in food based on nuclear magnetic resonance technology.

[0005] The present invention proposes a rapid qualitative identification method for sildenafil compounds in food based on nuclear magnetic resonance technology, which comprises: Collect standard spectra of sildenafil compounds, process them with topspin / CMC-assist software, and then import them into AssureNMR to establish a standard NMR spectrum library of sildenafil compounds. The sildenafil sample spectrum to be tested was imported and simulated matched with the database results.

[0006] According to the rapid qualitative identification method of the above-mentioned sildenafil compounds provided by the present invention, the sildenafil compound standard sample spectrum is first processed by topspin / CMC-assist software for data acquisition, and the data is Fourier transformed and then automatically and manually phase corrected and baseline calibrated. The HSQC spectra of the obtained multiple sildenafil compounds are superimposed and compared, and then the AssureNMR software tool is used for data processing to establish a sildenafil substance standard nuclear magnetic spectrum library. Finally, the sildenafil sample spectrum to be tested is imported into the AssureNMR software, and the current data is processed using the Amix window.

[0007] The nuclear magnetic resonance hydrogen spectrum of sildenafil standard is as follows Figure 1 shown.

[0008] The nuclear magnetic resonance hydrogen spectrum of pretreated sildenafil is as follows Figure 2 shown.

[0009] Several characteristic chemical shifts in the hydrogen spectrum of sildenafil-like substances are as follows: Figure 3 shown.

[0010] The quantile chart of the sildenafil substance library is as follows Figure 4 shown.

[0011] Specifically, the characteristic chemical shifts selected from the standard nuclear magnetic spectrum library of sildenafil-like substances are as follows: .

[0012] The spectrum of the sample to be tested is imported into the AssureNMR software. If the above characteristic peaks exist in the sample to be tested at the same time, it means that the sample to be tested contains sildenafil-like substances.

[0013] In some embodiments of the present invention, the preparation process of the sildenafil compound standard sample includes: weighing the sildenafil compound standard sample and dissolving it with DMSO-d6, and then transferring it to a nuclear magnetic resonance tube for nuclear magnetic resonance analysis.

[0014] Specifically, the preparation process of the sildenafil compound standard sample includes: weighing 5.0 mg of the sildenafil compound standard sample and dissolving it with 500 μL of DMSO-d6, and then transferring it to a nuclear magnetic resonance tube for nuclear magnetic resonance analysis.

[0015] In some embodiments of the present invention, the sildenafil compound standard sample includes at least sildenafil, narmosildenafil, homosildenafil, desmethylsildenafil, erythrodenafil, erythrodenafil, vardenafil, carbadenafil, phenylpropylcarbadenafil, sildenafil dimer, thio-edenafil and O-propyl-desmethylcarbadenafil.

[0016] In some embodiments of the present invention, the preparation process of the sildenafil test sample includes: accurately weighing the test sample into a plastic stoppered centrifuge tube, adding an organic solvent, ultrasonically extracting and centrifuging the sample, taking the supernatant, concentrating it under reduced pressure, dissolving it with DMSO-d6, and transferring it to a nuclear magnetic resonance tube for nuclear magnetic resonance analysis.

[0017] Specifically, the preparation process of the sildenafil test sample includes: accurately weighing 5.0 g of the test sample into a 15 mL plastic stoppered centrifuge tube, adding 10 mL of an organic solvent, ultrasonically extracting the sample for 5 minutes, centrifuging the sample at 4000 rpm for 5 minutes, taking the supernatant, concentrating it under reduced pressure, dissolving it with 500 μL of DMSO-d6, and transferring it to a nuclear magnetic resonance tube for nuclear magnetic resonance analysis; Preferably, the organic solvent is selected from dichloromethane, dichloroethane or methanol, more preferably dichloromethane. Extracting the sample with DCM can reduce interference from some water-soluble impurities or baselines and improve sample resolution.

[0018] In some embodiments of the present invention, the nuclear magnetic resonance measurement is performed using a Bruker Avance III HD 600 MHz Fourier transform superconducting nuclear magnetic resonance spectrometer.

[0019] In some embodiments of the present invention, the main parameters of the Bruker Avance III HD 600 MHz Fourier transform superconducting nuclear magnetic resonance spectrometer are as follows: Lock temperature: 298K; Time domain points: 64K; Pulse sequence: zg; spectral width: 20ppm; Scan times: 16 times; Spectrum center: 6.175ppm; Number of empty sweeps: 2 times; relaxation time: 1.0s.

[0020] In some embodiments of the present invention, the main parameter information of the standard nuclear magnetic spectrum library of sildenafil-like substances is as follows: .

[0021] In some embodiments of the present invention, standard data is imported and a classification matching method is established with the following parameters: (1) Classification method: k-nearest neighbor algorithm; (2) Classification options: Pareto scaling; minimum explained variance -95%; number of neighbors; (3) Confusion matrix: shows multiple / no assignment; minimum assignment probability - 95%; (4) Cross-validation: set aside 1% of the model table as the mode; Monte Carlo step -1.

[0022] In some embodiments of the present invention, the structure of sildenafil-like substances can be identified by a matching analysis method; the analysis method includes a k-nearest neighbor algorithm, a PLS analysis method, a SIMCA analysis method, or a PCA analysis method.

[0023] The present invention has at least the following beneficial effects: (1) After the database of some sildenafil-like substances is completed, subsequent testing does not require standard samples of a specific substance, saving a lot of money on purchasing standard samples and waiting time. In addition, rapid testing and identification can be performed without standard samples, effectively deterring criminals from modifying compound groups to evade supervision.

[0024] (2) Due to the high sensitivity of hydrogen atom abundance, the nuclear magnetic resonance hydrogen spectrum has a shorter scanning time than the two-dimensional spectrum, which significantly reduces the time and labor costs and makes the detection faster and more rapid.

[0025] (3) Nuclear magnetic resonance uses non-destructive testing technology, which facilitates sample recovery and subsequent experiments.

[0026] (4) Use different models and multiple sample references to support the matching results and reduce the risk of misjudgment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 is a hydrogen nuclear magnetic resonance spectrum of the sildenafil standard provided by the present invention; Figure 2 is a hydrogen nuclear magnetic resonance spectrum of the pretreated sildenafil provided by the present invention; Figure 3 These are several characteristic chemical shift patterns in the hydrogen spectrum of sildenafil-like substances provided by the present invention; Figure 4 is a quantile graph of the sildenafil-related substance library of the present invention; Figure 5 It is the actual sample classification matching result graph; Figure 6 This is the SIMCA verification result diagram of actual sample classification. DETAILED DESCRIPTION

[0029] Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention. The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0030] Example 1 Five health foods of different brands (sample 1, sample 2, sample 3, sample 4, sample 5) were purchased and tested to see whether they contained sildenafil-like substances.

[0031] Preparation of sample solution: Accurately weigh 5.0 g of the sample to be tested into a 15 mL plastic stoppered centrifuge tube, add 10 mL of dichloromethane, and extract by ultrasonication for 5 minutes. Centrifuge the sample at 4000 rpm for 5 minutes. Take the supernatant, concentrate it under reduced pressure, dissolve it in 500 μL of DMSO-d6, and transfer it to an NMR tube for testing.

[0032] The main parameters of the Fourier transform superconducting nuclear magnetic resonance spectrometer are as follows: Lock temperature: 298K; Time domain points: 64K; Pulse sequence: zg; spectral width: 20ppm; Scan times: 16 times; Spectrum center: 6.175ppm; Number of empty sweeps: 2 times; relaxation time: 1.0s.

[0033] The spectra were compared using AssureNMR software, and the results were as follows: Figure 5 As shown in the figure, it can be seen that in the actual sample classification matching results, an unknown positive sample was correctly classified into group Y. The SIMCA verification results of the sample classification are as follows: Figure 6 As shown, it is consistent with the sample model and is judged to be a sildenafil-like substance detected.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A rapid qualitative identification method for sildenafil compounds in food based on nuclear magnetic resonance technology, characterized in that: include: Collect standard spectra of sildenafil compounds, process them with topspin / CMC-assist software, and then import them into AssureNMR to establish a standard NMR spectrum library of sildenafil compounds. The sildenafil sample spectrum to be tested was imported and simulated matched with the database results.

2. The method according to claim 1, characterized in that The chemical shifts selected from the standard nuclear magnetic spectrum library of sildenafil-like substances are as follows: 。 3. The method according to claim 1, characterized in that The preparation process of the sildenafil compound standard sample includes: weighing the sildenafil compound standard sample, dissolving it with DMSO-d6, and then transferring it to a nuclear magnetic resonance tube for nuclear magnetic resonance measurement.

4. The method according to claim 1 or 3, characterized in that The sildenafil compound standard sample at least includes sildenafil, narmosildenafil, homosildenafil, desmethylsildenafil, erythrodenafil, narerythrodenafil, vardenafil, carbadenafil, phenylpropylcarbadenafil, sildenafil dimer, thio-eldenafil and O-propyl-desmethylcarbadenafil.

5. The method according to claim 1, characterized in that: The preparation process of the sildenafil test sample includes: accurately weighing the test sample into a plastic stoppered centrifuge tube, adding an organic solvent, ultrasonically extracting and centrifuging the sample, taking the supernatant, concentrating it under reduced pressure, dissolving it with DMSO-d6, and transferring it to a nuclear magnetic resonance tube for nuclear magnetic resonance analysis; Preferably, the organic solvent is selected from dichloromethane, dichloroethane or methanol, more preferably dichloromethane.

6. The method according to claim 3 or 5, characterized in that: The nuclear magnetic resonance measurement was performed using a Bruker Avance IIIHD 600 MHz Fourier transform superconducting nuclear magnetic resonance spectrometer.

7. The method according to claim 6, characterized in that The main parameters of the Bruker Avance III HD 600 MHz Fourier transform superconducting nuclear magnetic resonance spectrometer are as follows: Lock temperature: 298K; Time domain points: 64K; Pulse sequence: zg; spectral width: 20ppm; Scan times: 16 times; Spectrum center: 6.175ppm; Number of empty sweeps: 2 times; relaxation time: 1.0s.

8. The method according to claim 1, characterized in that: The main parameter information of the standard nuclear magnetic spectrum library of sildenafil-like substances is as follows: Number of spectra: 17; Group name: XDNF; Bucketing method: rectangular bucketing; Number of groups: 1; Integration mode: sum of all intensities; bucket range: 12.5ppm-0.5ppm; Zoom mode: Zoom to the total integral of all buckets; Bucket width: 0.01ppm.

9. The method according to claim 1, characterized in that: Import standard data and establish a classification matching method with the following parameters: (1) Classification method: k-nearest neighbor algorithm; (2) Classification options: Pareto scaling, minimum explained variance -95%, number of neighbors; (3) Confusion matrix: showing multiple / no assignments, minimum assignment probability - 95%; (4) Cross-validation: set aside 1% of the model table as the mode, Monte Carlo step -1.

10. The method according to claim 1, characterized in that: The analytical method can be matched to identify the structure of sildenafil-like substances; The analysis method includes a k-nearest neighbor algorithm, a PLS analysis method, a SIMCA analysis method or a PCA analysis method.

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