CN111982882B - A method for simultaneous rapid detection of carbendazim and thiophanate-methyl residues in tobacco - Google Patents

Abstract

The present invention provides a method for simultaneously rapidly detecting carbendazim and thiophanate-methyl residues in tobacco, comprising the following steps: (1) taking a tobacco sample and adding organic reagents, after ultrasonic vibration, transferring the liquid to a new centrifuge tube, After adding the purifying agent, vortexing and centrifuging, take the organic layer to dry, add the reconstituted solution to redissolve, and after layering, obtain the upper layer of carbendazim to be tested, and the lower layer of thiophanate-methyl to be tested; (2) All the After the liquid to be tested, the agglomerating agent and the gold nano-sol are evenly mixed, it is detected by a portable Raman spectrometer, and the measured surface-enhanced Raman spectrum is analyzed to obtain the detection results of carbendazim and thiophanate-methyl.

Description

A method for simultaneous rapid detection of carbendazim and thiophanate-methyl residues in tobacco

technical field

The invention relates to a detection method for pesticides carbendazim and thiophanate-methyl, in particular to the detection of residual carbendazim and thiophanate-methyl in tobacco by using surface-enhanced Raman spectroscopy, belonging to the field of pesticide residue analysis and detection.

Background technique

Carbendazim and thiophanate-methyl both belong to benzimidazoles and are a class of fungicides with high efficiency, broad spectrum and low toxicity. They play a bactericidal effect by interfering with the formation of spindles in the mitosis of pathogens and affecting cell division. It is widely used in the control of plant fungal diseases in agricultural production. Thiophanate-methyl is a pesticide registered in tobacco, which can be metabolized into carbendazim in plants. The study found that thiophanate-methyl has genotoxicity and reproductive toxicity, and its degradation or metabolite carbendazim has a long residual effect period and accumulated toxicity. Given the widespread application of carbendazim and thiophanate-methyl and their potential dose-dependent risks, it is important to develop rapid detection of carbendazim and thiophanate-methyl residues.

At present, the detection methods of carbendazim and thiophanate-methyl residues are mainly liquid chromatography and its combination with mass spectrometry, immunoassay, etc. For example, invention patent 200610097540.1 and invention patent 201710608203.2 respectively disclose liquid chromatography detection methods for carbendazim and thiophanate-methyl residues in vegetables and Dendrobium officinale. Patent 201410486081.0 discloses a method for simultaneous detection of pesticide residues such as carbendazim and thiophanate-methyl in citrus by LC-MS. This type of method has the advantages of high detection sensitivity and strong specificity, but often requires complex pre-treatment processes such as extraction and purification, has a long cycle, high cost, and requires expensive large-scale instruments, which is not suitable for on-site rapid detection of batch samples. Patent 201710250425.1 discloses a two-in-one enzyme-linked immunosorbent assay kit for the detection of carbendazim and thiophanate-methyl, which has the advantages of being quick and easy compared to traditional instrumental analysis techniques, but in obtaining high titer and high specificity antibodies, As well as improving the detection sensitivity and detection limit of the method, further research and improvement are still needed. Moreover, the structures of carbendazim and thiophanate-methyl are similar, and the current rapid detection methods still cannot solve the problem of mutual interference between them. Therefore, it is of great practical significance to seek a detection method for carbendazim and thiophanate-methyl with strong specificity, low cost, simple operation and fast detection speed.

Surface-enhanced Raman spectroscopy (SERS) is a very effective tool for detecting intermolecular interactions and characterizing surface molecular adsorption behavior and molecular structure. The advantages of carrying and low detection cost have attracted more and more attention in the rapid detection of environmental pollutants, food additives, and pesticide residues.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for simultaneous and rapid detection of carbendazim and thiophanate-methyl in tobacco based on surface-enhanced Raman spectroscopy.

In order to achieve the above object of the invention, the present invention provides a method for rapidly detecting carbendazim and thiophanate-methyl residues in tobacco simultaneously, comprising the following steps:

(1) Take a tobacco sample and add an organic reagent, after ultrasonic vibration, transfer the liquid to a new centrifuge tube, add a purifying agent, vortex vibration and centrifuge, take the organic layer to dry, add a reconstituted solution to reconstitute, and layer to obtain The upper layer of carbendazim to be tested, the lower layer of thiophanate-methyl to be tested;

Wherein, in order to fully dissolve carbendazim and thiophanate-methyl, the organic solvent is a mixed solvent of acetonitrile and toluene; the complex solution is a mixed solvent of cyclohexane and pure water,

In order to remove the interference of pigments, polyphenols, etc. in tobacco, the purifying agent is N-propylethylenediamine bonded solid phase adsorbent (PSA) and anhydrous magnesium sulfate;

(2) After mixing the liquid to be tested with the agglomerating agent and the nano-gold sol evenly, it is detected by a portable Raman spectrometer, and the measured surface-enhanced Raman spectrum is analyzed to obtain carbendazim and thiophanate-methyl. Test results.

In some embodiments, in step (1), in order to fully dissolve carbendazim and thiophanate-methyl, the tobacco sample is mixed with an organic reagent in a ratio of 1:5 to 20, and then ultrasonically oscillated for 1 to 5 minutes. The organic solvent is a mixed solvent of acetonitrile and toluene with a volume ratio of 5:0.5-2.5.

In some embodiments, in order to remove the interference of pigments, polyphenols, etc. in tobacco, in step (1), N-propylethylenediamine bonded solid phase adsorbent (PSA) and anhydrous magnesium sulfate are used as scavengers, The dosage of the PSA in each gram of tobacco sample is 0-0.1 g, the dosage of anhydrous magnesium sulfate is 0.1-1 g, vortexed for 1-3 min, and centrifuged at 8000-10000 r/min for 1-2 min.

In some embodiments, in step (1), the reconstituted solution is a mixed solvent of cyclohexane and pure water in a volume ratio of 1:1 to 3. After adding the reconstituted solution, vortex for 1 to 3 minutes, and the mixture is heated for 8000 to 10000 r. /min centrifugation for 1 to 2 minutes to obtain the sample liquid to be tested; wherein, the upper layer is the liquid to be tested for carbendazim, and the lower layer is the liquid to be tested for thiophanate-methyl.

In some embodiments, in order to adjust the ionic strength of the system and enable the gold nanoparticles to obtain the optimal enhancement ability, in step (2), the agglomeration agent is sodium chloride, potassium carbonate, potassium bromide, potassium chloride, sulfuric acid One or more of sodium, magnesium sulfate, potassium iodide are mixed, preferably, the agglomeration agent used in the upper carbendazim solution to be tested is 1mol/L potassium carbonate and 1mol/L potassium bromide aqueous solution by volume 1:2～5 Mixing, the agglomeration agent used in the lower thiophanate-methyl solution to be tested is 0.5-1.5 mol/L sodium chloride aqueous solution;

In some embodiments, in step (2), the particle size of the nano-gold sol particles is 20-150 nm, in order to ensure the uniformity of the size and morphology of the synthesized nano-gold sol and the stability of the nano-gold sol system, and comprehensively consider the system For the enhancement ability of the gold nano-sol, preferably, the particle size of the nano-gold sol is 50-80 nm.

In some embodiments, in step (2), the volume ratio of the liquid to be tested, agglomeration agent, and nano-gold sol is 1:0.1-1:0.8-6; preferably, the liquid to be tested, agglomeration agent, and gold nanoparticles The volume ratio of the sol is 1:0.2-0.6:1-4.

In some embodiments, in step (2), the parameters of the portable Raman spectrometer are an excitation light source of 785 nm, an excitation power of 50-500 mW, and a scanning time of 100-10000 ms. Preferably, the excitation power is 500 mw and the scanning time is 5000 ms.

In some embodiments, in step (2), the collected sample surface-enhanced Raman spectrum is compared with the surface-enhanced Raman spectrum of carbendazim and thiophanate-methyl standard products, wherein the surface of carbendazim is Enhanced Raman peaks: 625cm ^-1 , 756cm ^-1 , 903cm ^-1 , 940cm ^-1 , 1006cm ^-1 , 1082cm ^-1 , 1224cm ^-1 , 1263cm ^-1 ; surface enhanced Raman peaks of thiophanate-methyl : 605cm ^-1 , 712cm ^-1 , 961cm ^-1 , 1039cm ^-1 , 1189cm ^-1 , 1259cm ^-1 ; according to the characteristic peaks, it can be judged whether the sample contains carbendazim or thiophanate-methyl.

Compared with the existing detection methods, the method for simultaneous and rapid detection of carbendazim and thiophanate-methyl residues in tobacco provided by the present invention has the obvious advantage of strong specificity. This method separates carbendazim and thiophanate-methyl in different solvent layers of the same treatment solution through simple pretreatment, and combines the appropriate agglomerating agent system and the fingerprint identification advantages of surface-enhanced Raman spectroscopy to realize the rapid detection method. Specific recognition of thiophanate-methyl and carbendazim.

In addition, the method provided by the present invention has simple pretreatment steps for tobacco samples, convenient operation, fast efficiency, low cost, low consumption of organic reagents, and does not need to rely on large-scale equipment, and is suitable for rapid screening of on-site batch samples.

Description of drawings

Figure 1 shows the surface-enhanced Raman spectra of solid Raman spectra of carbendazim and standard solutions of different concentrations, where a is the gold sol blank, and b, c, d, and e are 0.01, 0.05, 0.1, and 0.2 mg/L polybacterium, respectively. is the surface-enhanced Raman spectrum of the standard solution of carbendazim, and f is the solid Raman spectrum of carbendazim;

Figure 2 shows the surface-enhanced Raman spectrum of thiophanate-methyl solid and standard solutions of different concentrations, where a is the gold sol blank, and b, c, d, and e are 0.01, 0.05, 0.1, and 0.2 mg/L, respectively. Surface-enhanced Raman spectrum of thiophanate-methyl standard solution, f is the solid Raman spectrum of thiophanate-methyl;

Figure 3 is the surface-enhanced Raman spectrum of the tobacco sample containing carbendazim, wherein a, b, and c are the surface-enhanced Raman spectrum of the tobacco sample extract containing 0, 1, and 4 mg/kg of carbendazim, respectively, and d is the surface-enhanced Raman spectrum of the carbendazim-containing tobacco sample. Spirit standard surface-enhanced Raman spectroscopy;

Fig. 4 is the surface-enhanced Raman spectrum of the tobacco sample containing thiophanate-methyl, wherein a, b, and c are the surface-enhanced Raman spectrum of the tobacco sample extract containing 0, 2, and 5 mg/kg thiophanate-methyl, respectively, d is the surface-enhanced Raman spectrum of the standard thiophanate-methyl;

Fig. 5 is the surface-enhanced Raman spectrum of tobacco samples containing carbendazim and thiophanate-methyl simultaneously, wherein a is the detection result of the upper layer of tobacco sample No. 1 to be tested, and b is the detection result of the upper layer of tobacco sample of No. 2 to be tested, c is the detection result of the liquid to be tested in the lower layer of the No. 1 tobacco sample, and d is the detection result of the liquid to be tested in the lower layer of the No. 2 tobacco sample.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and do not limit the protection scope of the present invention

Preparation of nano-gold sol

Boil 200 mL of chloroauric acid solution with a concentration of 0.01 wt %, and then quickly add 1.5 mL of sodium citrate solution (with a concentration of 1 wt %) to it at one time. The solution turns reddish-brown in about 3 minutes, keeps boiling for 30 minutes, and cools for later use.

Raman spectrum collection of carbendazim standard

After the standard carbendazim solid powder was placed on a clean glass slide and flattened, the Raman spectrum was collected on a portable Raman spectrometer to obtain the Raman spectrum shown in Figure 1.

Prepare standard aqueous solutions of carbendazim with concentrations of 0.2, 0.1, 0.05, 0.01, and 0 mg/L, respectively, take 100 μL of the standard solution, and add 50 μL of agglomeration agent (1mol/L potassium carbonate and 1mol/L potassium bromide aqueous solution in a volume ratio of 1: 4 mixing), add 300 μL of gold nanosol, and collect surface-enhanced Raman spectra with a portable Raman spectrometer under the conditions of excitation light of 785 nm, laser power of 500 mW, and scanning time of 5000 ms. The results are shown in Figure 1. Comparing the Raman spectra of carbendazim solid powder and gold sol blank, it can be seen that the characteristic peaks of carbendazim are 625cm ^-1 , 756cm ^-1 , 903cm ^-1 , 940cm ^-1 , 1006cm ^-1 , 1082cm ^-1 , 1224cm ^-1 , 1263cm ^-1 . The results show that the detection concentration of carbendazim standard solution can reach 0.01mg/L.

Raman spectrum collection of thiophanate-methyl standard

After the standard thiophanate-methyl solid powder was placed on a clean glass slide and flattened, the Raman spectrum was collected on a portable Raman spectrometer to obtain the Raman spectrum shown in Figure 2.

Prepare standard aqueous solutions of thiophanate-methyl with concentrations of 0.2, 0.1, 0.05, 0.01, and 0 mg/L, respectively, take 200 μL of standard solution, add 50 μL of agglomeration agent (1 mol/L aqueous sodium chloride solution), and add 200 μL of gold nanosol. Under the conditions of excitation light of 785 nm, laser power of 500 mW, and scanning time of 5000 ms, the surface-enhanced Raman spectrum was collected with a portable Raman spectrometer. The results are shown in Figure 2. Comparing the Raman spectra of thiophanate-methyl solid powder and gold sol blank, it can be seen that the characteristic peaks of thiophanate-methyl are 605cm ^-1 , 712cm ^-1 , 961cm ^-1 , 1039cm ^-1 , 1189cm ^-1 , 1259cm ^-1 . The results show that the detection concentration of thiophanate-methyl standard solution can reach 0.01mg/L by this detection method.

Embodiment 1. Detection of tobacco samples containing carbendazim

0.5g of tobacco samples were weighed respectively (the contents of carbendazim were measured by liquid chromatography-mass spectrometry and were respectively 0, 1, and 4 mg/kg), and 4 mL of organic reagents (acetonitrile and toluene were mixed at a ratio of 5:1) were added with ultrasonic vibration. 2min, take the supernatant into a new centrifuge tube, add 0.2g anhydrous magnesium sulfate and 0.03g PSA purifying agent, vortex for 2min, and centrifuge at 8000r/min for 2min, take 3mL of the organic layer and blow dry with nitrogen, add 500μL of purified water and 250 μL of cyclohexane, vortexed for 2 min, and centrifuged at 8000 r/min for 2 min, the upper organic layer is the liquid to be tested. Take 100 μL of the solution to be tested, add 50 μL of agglomeration agent (mixing 1 mol/L potassium carbonate and 1 mol/L potassium bromide aqueous solution in a volume ratio of 1:4), add 300 μL of nano-gold sol and mix evenly, under excitation light source 785 nm, laser power 500 mW, Under the condition of scanning time of 5000ms, the Raman spectrum of the sample was collected with a portable Raman spectrometer, and the results are shown in Figure 3.

Embodiment two, containing thiophanate-methyl tobacco sample detection

Weigh 0.5g of tobacco sample respectively (wherein the content of thiophanate-methyl, measured by liquid chromatography-mass spectrometry, is respectively 0, 2, 5mg/kg), add 4mL of organic reagent (acetonitrile and toluene are mixed in a ratio of 5:1) Ultrasonic vibration for 2 min, take the supernatant into a new centrifuge tube, add 0.25 g anhydrous magnesium sulfate and 0.04 g PSA purifying agent, vortex for 2 min, and centrifuge at 8000 r/min for 2 min, take 3 mL of the organic layer and dry it with nitrogen, add 500 μL Purified water and 200 μL of cyclohexane, vortexed for 2 min, and centrifuged at 8000 r/min for 2 min, the lower water layer was the liquid to be tested. Take 200 μL of the liquid to be tested, add 50 μL of agglomeration agent (1mol/L sodium chloride aqueous solution), add 200 μL of nano-gold sol and mix evenly, under the conditions of excitation light source 785nm, laser power 500mW, and scanning time 5000ms, collect with a portable Raman spectrometer Raman spectrum of the sample. The results are shown in Figure 4.

Embodiment three, contain carbendazim and thiophanate-methyl simultaneously to detect tobacco leaf samples

Weigh 0.5g of tobacco samples respectively (the contents of carbendazim and thiophanate-methyl in sample No. 1 were measured by liquid chromatography-mass spectrometry and were 2 mg/kg and 9 mg/kg respectively; Thiophanate content was 4mg/kg, 6mg/kg respectively), add 4mL organic reagent (acetonitrile and toluene are mixed at a ratio of 5:1), ultrasonically shake for 2min, take the supernatant in a new centrifuge tube, add 0.3g anhydrous Magnesium sulfate and 0.03g PSA purifying agent, vortexed for 2min, centrifuged at 8000r/min for 2min, took 3mL of organic layer to dry with nitrogen, added 500μL of purified water and 250μL of cyclohexane, vortexed for 2min, and centrifuged at 8000r/min for 2min Afterwards, the upper organic layer and the lower water layer were processed according to the methods of Example 1 and Example 2 respectively, the upper organic layer was detected with carbendazim, and the lower water layer was detected with thiophanate-methyl. The results are shown in Figure 5.

The above-mentioned specific embodiments describe in detail the technical solutions and beneficial effects of the present invention. It should be understood that the above-mentioned embodiments are only the most preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, additions and equivalent substitutions made within the scope shall be included within the protection scope of the present invention.

Claims (12)

1. a method for rapidly detecting carbendazim and thiophanate-methyl residues in tobacco simultaneously, is characterized in that, this method may further comprise the steps: (1) Take a tobacco sample and add an organic solvent, after ultrasonic vibration, transfer the liquid to a new centrifuge tube, add a purifying agent, vortex vibration and centrifuge, take the organic layer to dry, add a reconstituted solution to reconstitute, and layer to obtain The upper layer of carbendazim to be tested, the lower layer of thiophanate-methyl to be tested; Wherein, the organic solvent is a mixed solvent of acetonitrile and toluene, and the complex solution is a mixed solvent of cyclohexane and pure water, Described purifying agent is N-propyl ethylenediamine bonded solid phase adsorbent PSA and anhydrous magnesium sulfate; (2) After mixing the liquid to be tested with the agglomerating agent and nano-gold sol evenly, it is detected by a portable Raman spectrometer, and the measured surface-enhanced Raman spectrum is analyzed to obtain carbendazim and thiophanate-methyl. Test results. 2. The method according to claim 1, wherein In step (1), after mixing the tobacco sample with an organic solvent in a ratio of 1:5-20, ultrasonically oscillating for 1-5 min, the organic solvent is a mixed solvent of acetonitrile and toluene with a volume ratio of 5:0.5-2.5; The dosage of the PSA in each gram of tobacco sample is 0-0.1 g, the dosage of anhydrous magnesium sulfate is 0.1-1 g, vortex shaking for 1-3 min, and centrifugation at 8000-10000 r/min for 1-2 min. 3 . The method according to claim 1 , wherein in step (1), the reconstituted solution is a mixed solvent of cyclohexane and pure water in a volume ratio of 1:1 to 3, and after adding the reconstituted solution, vortex oscillates. 4 . 1-3 min, and centrifuged at 8,000-10,000 r/min for 1-2 min to obtain the sample solution to be tested; the upper layer is the solution to be tested for carbendazim, and the lower layer is the solution to be tested for thiophanate-methyl. 4. The method according to claim 1, wherein in step (2), the agglomeration agent is sodium chloride, potassium carbonate, potassium bromide, potassium chloride, sodium sulfate, magnesium sulfate, potassium iodide one or a mixture of several. 5. method according to claim 4, is characterized in that, the used agglomeration agent of upper layer carbendazim liquid to be tested is that 1 mol/L potassium carbonate and 1 mol/L potassium bromide aqueous solution mix by volume ratio 1:2～5 , the agglomeration agent used in the lower thiophanate-methyl test solution is 0.5-1.5 mol/L sodium chloride aqueous solution. 6 . The method according to claim 1 , wherein in step (2), the particle size of the nano-gold sol particles is 20-150 nm. 7 . 7 . The method according to claim 6 , wherein the particle size of the nano-gold sol particles is 50-80 nm. 8 . 8 . The method according to claim 1 , wherein in step (2), the volume ratio of the liquid to be tested, the agglomerating agent, and the nano-gold sol is 1:0.1-1:0.8-6. 9 . The method according to claim 1, wherein in step (2), the parameters of the portable Raman spectrometer are an excitation light source of 785 nm, an excitation power of 50-500 mW, and a scanning time of 100-10000 ms. 10 . The method according to claim 9 , wherein the excitation power is 500 mw and the scanning time is 5000 ms. 11 . The method according to claim 1, wherein in step (2), the collected sample surface-enhanced Raman spectrum and the surface-enhanced Raman spectrum of carbendazim and thiophanate-methyl standard products For comparison, the surface-enhanced Raman characteristic peaks of carbendazim are: 625 cm ^-1 , 756 cm ^-1 , 903 cm ^-1 , 940 cm ^-1 , 1006 cm ^-1 , 1082 cm ^-1 , 1224 cm ^-1 , 1263 cm ^-1 ; surface-enhanced Raman characteristic peaks of thiophanate-methyl: 605 cm ^-1 , 712 cm ^-1 , 961 cm ^-1 , 1039 cm ^-1 , 1189 cm ^-1 , 1259 cm ^-1 ; according to characteristic peaks It can be judged whether the sample contains carbendazim or thiophanate-methyl. 12 . The method according to claim 8 , wherein the volume ratio of the liquid to be tested, the agglomerating agent, and the nano-gold sol is 1:0.2-0.6:1-4. 13 .

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