CN115184446A - Method for detecting paclobutrazol in aconite root soil and/or different parts of aconite root - Google Patents

Abstract

The invention belongs to the technical field of quality detection, in particular to a method for detecting paclobutrazol in aconite soil and/or different parts of aconite. The technical problem to be solved by the present invention is to provide a method for detecting paclobutrazol in aconite soil and/or different parts of aconite, and real-time monitoring the distribution of paclobutrazol in soil and different parts during the growth of aconite through high-resolution mass spectrometry imaging. The method of the invention is as follows: a. preparing the solution of the sample to be tested and the solution of the reference substance; b. punching a hole on the filter paper, respectively drawing the solution of the sample to be tested and the solution of the reference substance for spotting, drying it, and placing it on a conductive glass plate; c. . Using DESI to detect the spotting samples, perform mass spectrometry imaging scanning, and determine the residual paclobutrazol in the samples to be tested according to the imaging results. The method of the invention can be more rapid and intuitive, and the distribution of paclobutrazol in different parts of aconite and the difference of the content of paclobutrazol in different parts can be reflected through the imaging map.

Description

Method for detecting paclobutrazol in aconite soil and/or different parts of aconite

technical field

The invention belongs to the technical field of quality detection, in particular to a method for detecting paclobutrazol in aconite soil and/or different parts of aconite.

Background technique

Paclobutrazol is a triazole plant growth regulator, which can delay plant growth, improve lodging resistance and stress resistance, and increase crop yield. Paclobutrazol has been widely used in the cultivation of crops, vegetables, fruit trees, etc., and is gradually being used in the cultivation of Chinese medicinal materials. For example, during the cultivation of Ophiopogon japonicus, through the long-term use of plant growth regulators such as paclobutrazol in large quantities, the existing dosage of paclobutrazol is more than 100 times that of rice. However, excessive use of paclobutrazol not only easily leads to soil compaction, but also reduces the quality of medicinal materials, and has certain toxicity.

my country has formulated the maximum residue limit standard (GB2763-2016) for some plant growth regulators. Most of the products involved in the limit standard are fruits, vegetables, etc., and Chinese medicinal materials are not involved. Most of the literature reports are also for the detection of plant growth regulator residues in fruits and vegetables, and few literatures involve the detection of plant growth regulator residues in Chinese medicinal materials. When collecting aconite samples, there is often a huge difference in the size and size of aconite. Therefore, it is very necessary to monitor paclobutrazol during the growth of Aconite, in order to provide a reference for the quality of Aconite medicinal materials, and also help to ensure the safety of medicinal materials.

There are many detection methods for paclobutrazol, such as gas chromatography mass spectrometry, high performance liquid chromatography, liquid chromatography mass spectrometry and the like. These methods are mainly for the detection of medicinal materials, but the mass spectrometry imaging detection of paclobutrazol in different medicinal parts of the soil and medicinal parts during the planting process of medicinal materials has not yet been applied, and there has been no application for the detection of paclobutrazol in different parts of aconite during the growth process of aconite. Method for the detection of paclobutrazol.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method that can quickly detect paclobutrazol in soil and/or different parts of aconite, and monitor the distribution of paclobutrazol in soil and different parts of aconite in real-time through high-resolution mass spectrometry imaging during the growth of aconite.

The technical scheme adopted by the present invention to solve the above technical problems is to provide a method for detecting paclobutrazol in aconite soil and/or different parts of aconite. The method includes the following steps:

a. Prepare the sample solution to be tested and the reference solution;

b. Punch holes on the filter paper, draw the sample solution to be tested and the reference solution for spotting, dry it, and place it on a conductive glass plate;

c. Use DESI to detect the spotting samples, perform mass spectrometry imaging scanning, and determine the residual paclobutrazol in the samples to be tested according to the imaging results.

Wherein, in the above-mentioned method for detecting paclobutrazol in aconite soil and/or different parts of aconite, in step b, the suction amount of the sample solution to be tested is 2-10 μL.

Further, in step b, the diameter of the holes punched in the filter paper is 4-10 mm.

Wherein, in the above method for detecting paclobutrazol in aconite soil and/or different parts of aconite, in step c, the DESI detection conditions are: positive ion full scan, spray voltage 3.5-3.9kV. Scanning range: 100～600m/z. Spatial resolution 110 ~ 200μm. Nebulizer pressure 0.6 ~ 0.8MPa. Ion source temperature 160 ~ 180 ℃.

Preferably, in step c, the DESI detection conditions are: positive ion full scan, spray voltage 3.5kV. Scanning range: 100～600m/z. Spatial resolution 110 μm. Nebulizer pressure 0.6MPa. The ion source temperature was 160°C.

Further, in step c, the atomizing agent used in the DESI detection is an aqueous solution containing 0.35-0.45 wt % formic acid and 98-100 wt % methanol.

Preferably, in step c, the atomizing agent used in the DESI detection is an aqueous solution containing 0.4 wt % formic acid and 98 wt % methanol.

Wherein, in the above-mentioned method for detecting paclobutrazol in different parts of aconite soil and/or aconite, in step a, the preparation of the sample solution to be tested is to dry the aconite root soil, aconite root and aconite stem and leaf in different growth stages, and powder , sieve, add glacial acetic acid solution to stand, add acetonitrile and mix well, add mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate, shake, cool, centrifuge, take the supernatant and place it in the dispersed solid phase containing the purification material Extract the purification tube to complete the purification, centrifuge, take the supernatant and place it on a nitrogen blower to concentrate in a water bath, add acetonitrile, mix well, and filter.

Preferably, in step a, the preparation of the sample solution to be tested is to dry aconite root soil, aconite roots and aconite stems and leaves in different growth stages, powder, pass through a No. 3 sieve, respectively accurately weigh 3g and place them in 50mL of polypropylene In a stoppered centrifuge tube, add 15 mL of 1% glacial acetic acid solution, vortex to fully infiltrate the powder, let stand for 30 min, add 15 mL of acetonitrile, vortex to mix, set the shaker for 5 min, add anhydrous magnesium sulfate and The mixed powder of anhydrous sodium acetate, wherein the mass ratio of anhydrous magnesium sulfate and anhydrous sodium acetate is 4:1, shake to disperse, shake for 3 minutes, cool in an ice bath for 10 minutes, centrifuge for 5 minutes, take 9 mL of supernatant and place it in Put the disperse solid phase extraction purification tube containing the purification material to complete the purification, centrifuge for 5 minutes, accurately draw 5 mL of the supernatant, place it on a nitrogen blower, and concentrate it to 4 mL in a water bath at 40 °C, add acetonitrile to dilute to 1.0 mL, and mix well. Just filter.

Further, in step a, the purification materials are 900 mg of anhydrous magnesium sulfate, 300 mg of N-propylethylenediamine, 300 mg of octadecylsilane-bonded silica gel, 300 mg of silica gel, and 90 mg of graphitized carbon black.

The beneficial effects of the present invention are:

The present invention adopts DESI (electrospray desorption ionization imaging technology) to perform mass spectrometry imaging on the root soil of aconite and/or paclobutrazol in different parts of aconite. Compared with the commonly used liquid chromatography method in the prior art, the method of the present invention can be more rapid and intuitive , the distribution of paclobutrazol in different parts of aconite and the content difference of paclobutrazol in different parts can be reflected by the imaging map, which can obtain more information than liquid chromatography. The method of the invention can realize the real-time monitoring of the plant growth regulator of the aconite during the growth process, so as to grasp the growth condition of the aconite.

Description of drawings

Figure 1 shows the spot distribution of the soil samples from the roots of Aconite and different parts of Aconite in Example 1.

FIG. 2 is a mass spectrometry image of paclobutrazol in the soil of aconite root and samples from different parts of aconite in Example 1. FIG.

Figure 3 shows the response values of paclobutrazol in the soil of the root of Aconite in Verification Example 1 and samples from different parts of Aconite.

Detailed ways

The present invention will be described in further detail below through specific embodiments.

Example 1

a. Preparation of test sample and reference solution

According to Table 1, collect the growth soil of Aconite at different time points, the aerial part (stem and leaf), and the underground part (root) of Aconite. sieve, accurately weigh 3g of the powder and place it in a 50mL polystyrene centrifuge tube with a stopper, add 15mL of 1% glacial acetic acid solution, vortex to fully infiltrate the powder, leave it for 30min, add 15mL of acetonitrile, vortex to mix well, and set it to shake Vigorously shake (500 times/min) for 5min on the machine, add the mixed powder of 6g anhydrous magnesium sulfate and 1.5g anhydrous sodium acetate, shake it up immediately, and then place it on the shaker to shake vigorously (500 times/min) for 3min. Cool in an ice bath for 10 min, centrifuge (4000 rpm) for 5 min, take 9 mL of the supernatant, and place it in a dispersive solid-phase extraction purification tube pre-installed with purification materials. The purification materials are anhydrous magnesium sulfate 900 mg, N-propyl 300 mg of ethylenediamine, 300 mg of octadecylsilane-bonded silica gel, 300 mg of silica gel, and 90 mg of graphitized carbon black, vortexed to mix well, placed on a shaker for vigorous shaking (500 times/min) for 5 min to complete purification, and centrifuged (4000 rpm) for 5 minutes, accurately aspirated 5 mL of supernatant, placed on a nitrogen blower and concentrated to 0.4 mL in a 40°C water bath, added acetonitrile to dilute to 1.0 mL, vortexed and mixed, filtered, and the subsequent filtrate was taken, namely have to.

Precisely weigh an appropriate amount of the paclobutrazol reference substance, dissolve it in methanol, and prepare a solution containing 1-1000 μg of paclobutrazol per 1 mL.

b. Spotting

Punch holes on the filter paper with a diameter of 4 mm, draw 2 μL of the sample solution to be tested and the reference solution for spotting, dry them, and place them on a conductive glass plate. The specific spotting sample information is shown in Figure 1.

c. Detection

The conductive glass plate was placed on the mass spectrometer imager for detection, and the positive ion full scan of the analytical electrospray ionization source (DESI), the spray voltage was 3.5kV, the scanning range: 100-600m/z, the spatial resolution was 110μm, and the sprayer pressure was 0.6MPa. The atomizing agent is an aqueous solution containing 0.4 wt % formic acid and 98 wt % methanol, and the temperature of the ion source is 160°C. According to the imaging results, the residue of paclobutrazol in the sample to be tested is judged.

The obtained raw data was analyzed by HDI software. The results are shown in Figure 2. Corresponding to Figure 1, the yellow color is the paclobutrazol reference substance, the light blue color is mainly the collected soil samples, and the other black samples represent Paclobutrazol was not detected.

Table 1 Sample source information table

Verification Example 1

The liquid chromatography-mass spectrometry method was used to detect paclobutrazol in aconite soil and different parts, including the following steps:

A, prepare test sample, reference substance solution, with step a in embodiment 1;

B. Instrumental analysis conditions; chromatographic conditions: chromatographic column: Acquity UPLC ^R BEH C18 (2.1mm×100mm, 1.7μm) chromatographic column; mobile phase: 0.1% ammonium formate water (A)-0.1% ammonium formate acetonitrile (B), The gradient elution procedure is shown in Table 2 below, the flow rate is 0.4 mL·min ^-1 , the column temperature is 40° C., and the injection volume is 1 μL.

Table 2 UPLC gradient elution program

Time (t/min) 0.1% ammonium formate aqueous solution (A) 0.1% Ammonium Formate Acetonitrile (B) 0 95 5 3 70 30 5 30 70 6 0 100 11 0 100 12 95 5 14 95 5

Q-TOF/MS conditions

Mass spectrometry was performed on a Waters SYNAPT G2 HDMS system. Using nitrogen as atomization, cone gas, source temperature: 150℃, reverse cone gas flow: 50L/h, desolvation temperature: 450℃, desolvation gas flow gas flow): 800L/h, Sampling cone: 40V, Extraction cone: 4V, Capillary voltage: 2.5kV in positive ion mode, Scan time: 0.2 s, Inter scan time: 0.02s, mass-to-charge ratio: m/z 100-600 Da, locked mass leucine enkephalin: positive ion mode (ESI+) m/z 556.2766.

C. Data analysis.

The collected samples were collected and analyzed by liquid chromatography-mass spectrometry. It was found that the collected samples contained a certain amount of paclobutrazol in the soil samples, and the content of paclobutrazol in individual samples above and below ground was higher. See Fig. 3 for details. The response value of each sample in the figure basically matches the response value of the above-mentioned mass spectrometry imaging.

It can be seen from the above examples that the method of the present invention is verified by combining mass spectrometry imaging and liquid chromatography-mass spectrometry, and the test results are basically consistent. By adopting the method of the invention, high-resolution mass spectrometry imaging of paclobutrazol in different parts of aconite planting soil and aconite can be realized, so that the paclobutrazol residue in the sample can be quickly and intuitively analyzed.

Claims (10)

1. the method for detecting paclobutrazol in different parts of aconite soil and/or aconite, is characterized in that: comprise the steps: a. Prepare the sample solution to be tested and the reference solution; b. Punch holes on the filter paper, draw the sample solution to be tested and the reference solution for spotting, dry it, and place it on a conductive glass plate; c. Use DESI to detect the spotting samples, perform mass spectrometry imaging scanning, and determine the residual paclobutrazol in the samples to be tested according to the imaging results. 2 . The method for detecting paclobutrazol in aconite soil and/or different parts of aconite according to claim 1 , wherein in step b, the amount of the sample solution to be tested is 2-10 μL. 3 . 3. The method for detecting paclobutrazol in aconite soil and/or different parts of aconite according to claim 1 or 2, characterized in that: in step b, the diameter of the holes punched on the filter paper is 4-10 mm. 4. the method for detecting paclobutrazol in aconite soil and/or different parts of aconite according to claim 1, is characterized in that: in step c, the condition that described DESI detects is: positive ion full scan, spray voltage 3.5～3.9kV ; Scanning range: 100～600m/z; Spatial resolution 110～200μm; Nebulizer pressure 0.6～0.8MPa; Ion source temperature 160～180℃. 5. the method for detecting paclobutrazol in aconite soil and/or aconite different parts according to claim 4, is characterized in that: in step c, the condition that described DESI detects is: positive ion full scan, spray voltage 3.5kV; Range: 100～600m/z; spatial resolution 110μm; nebulizer pressure 0.6MPa; ion source temperature 160℃. 6. the method for detecting paclobutrazol in aconite soil and/or different parts of aconite according to claim 1, is characterized in that: in step c, the atomizing agent that described DESI detects and adopts is formic acid containing 0.35～0.45wt% and 98-100 wt% methanol in water. 7. the method for detecting paclobutrazol in different parts of aconite soil and/or aconite according to claim 6, is characterized in that: in step c, the atomizer that described DESI detects and adopts is formic acid containing 0.4wt% and 98wt% methanol in water. 8. the method for detecting paclobutrazol in aconite soil and/or different parts of aconite according to claim 1, is characterized in that: in step a, the preparation of described sample solution to be tested is to separate the aconite root soil of different growth periods, Aconite roots and aconite stems and leaves were dried, powdered, sieved, added with glacial acetic acid solution and left to stand, added with acetonitrile and mixed well, added with the mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate, shaken, cooled, centrifuged, and the supernatant liquid was placed Complete the purification in a disperse solid phase extraction purification tube containing purification materials, centrifuge, take the supernatant and place it on a nitrogen blower to concentrate in a water bath, add acetonitrile, mix well, and filter. 9. the method for detecting aconite soil and/or paclobutrazol in different parts of aconite according to claim 8, is characterized in that: in step a, the preparation of described sample solution to be tested is to combine the aconite root soil, aconite in different growth periods The roots and aconite stems and leaves were dried, powdered, passed through a No. 3 sieve, and 3 g were accurately weighed and placed in a 50 mL polypropylene centrifuge tube with a stopper. Set for 30min, add 15mL of acetonitrile, vortex to mix, set the shaker to shake for 5min, add the mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate, wherein the mass ratio of anhydrous magnesium sulfate and anhydrous sodium acetate is 4:1, Shake up, shake for 3 min, cool in an ice bath for 10 min, centrifuge for 5 min, take 9 mL of supernatant and place it in a dispersive solid-phase extraction purification tube containing purification materials to complete purification, centrifuge for 5 minutes, and accurately draw 5 mL of supernatant Placed on a nitrogen blower and concentrated to 4 mL in a water bath at 40°C, diluted to 1.0 mL by adding acetonitrile, mixed well, and filtered to obtain. 10. the method for detecting paclobutrazol in different parts of aconite soil and/or aconite according to claim 8 or 9, is characterized in that: in step a, described purification material is anhydrous magnesium sulfate 900mg, N-propyl ethylenedi 300 mg of amine, 300 mg of octadecylsilane-bonded silica gel, 300 mg of silica gel, and 90 mg of graphitized carbon black.

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