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

Abstract

The present invention belongs to the field of quality inspection technology, and specifically relates to a method for detecting paclobutrazol in 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 soil and / or different parts of aconite, and to monitor the distribution of paclobutrazol in soil and different parts of aconite in real time during the growth of aconite by high-resolution mass spectrometry imaging. The method of the present invention comprises the following steps: a. preparing a test sample solution and a reference solution; b. punching holes in filter paper, respectively aspirating the test sample solution and the reference solution for spotting, drying, and placing on a conductive glass plate; c. using DESI to detect the spot sample, performing mass spectrometry imaging scanning, and determining the residual paclobutrazol in the test sample based on the imaging results. The method of the present invention is faster and more intuitive, and can reflect the distribution of paclobutrazol in different parts of aconite and the difference in the content of paclobutrazol in different parts through imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection, and particularly relates to a method for detecting paclobutrazol in aconite soil and / or different parts of aconite. Background Art

[0002] Paclobutrazol is a triazole plant growth regulator that can slow 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, and is gradually being used in the cultivation of Chinese medicinal materials. For example, in the cultivation of Ophiopogon japonicus, the long-term and large-scale use of plant growth regulators such as paclobutrazol has led to the current use of paclobutrazol reaching more than 100 times that of rice. However, excessive use of paclobutrazol not only easily leads to soil compaction, but also causes a decline in the quality of medicinal materials and has certain toxicity.

[0003] my country has established maximum residue limits (MRLs) for some plant growth regulators (GB2763-2016). These limits primarily cover fruits and vegetables, not traditional Chinese medicines. The vast majority of literature reports focus on the detection of PGR residues in fruits and vegetables, with few studies examining their presence in traditional Chinese medicines. Because aconite samples often vary greatly in size, monitoring paclobutrazol during their growth is crucial to provide a reference for quality assurance and ensure safety.

[0004] There are many existing methods for detecting paclobutrazol, such as gas chromatography-mass spectrometry, high-performance liquid chromatography, and liquid chromatography-mass spectrometry. These methods primarily target medicinal herbs, but have yet to be applied to the mass spectrometry imaging detection of paclobutrazol in the soil used to grow the herbs or in different medicinal parts during the herb cultivation process. Furthermore, there are no methods for detecting paclobutrazol in different parts of aconite during its growth process. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for quickly detecting paclobutrazol in aconite soil and / or different parts of aconite, and to monitor the distribution of paclobutrazol in the soil and different parts of aconite in real time during the growth of aconite by high-resolution mass spectrometry imaging.

[0006] The present invention solves the above-mentioned technical problems by providing a method for detecting paclobutrazol in aconite soil and / or different parts of aconite. The method comprises the following steps:

[0007] a. Prepare the sample solution and reference solution to be tested;

[0008] b. Punch a hole on the filter paper, draw the sample solution and the reference solution respectively, dry them, and place them on a conductive glass plate;

[0009] c. Use DESI to detect the spot sample and perform mass spectrometry imaging scanning. According to the imaging results, the residue of paclobutrazol in the sample to be tested is determined.

[0010] Wherein, in the above method for detecting paclobutrazol in aconite soil and / or different parts of aconite, in step b, the amount of the sample solution to be tested is 2 to 10 μL.

[0011] Furthermore, in step b, the diameter of the holes punched on the filter paper is 4 to 10 mm.

[0012] 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 3.5-3.9 kV, scan range: 100-600 m / z, spatial resolution 110-200 μm, sprayer pressure 0.6-0.8 MPa, and ion source temperature 160-180°C.

[0013] Preferably, in step c, the DESI detection conditions are: positive ion full scan, spray voltage 3.5 kV, scan range: 100-600 m / z, spatial resolution 110 μm, nebulizer pressure 0.6 MPa, and ion source temperature 160° C.

[0014] Furthermore, 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.

[0015] 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.

[0016] Wherein, in the above-mentioned method for detecting paclobutrazol in aconite soil and / or different parts of aconite, in step a, the preparation of the test sample solution is respectively drying the aconite root soil, aconite roots and aconite stems and leaves at different growth periods, pulverizing, sieving, adding glacial acetic acid solution and standing, adding acetonitrile and mixing, adding a mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate, oscillating, cooling, centrifuging, taking the supernatant and placing it in a dispersed solid phase extraction purification tube equipped with a purification material to completely purify it, centrifuging, taking the supernatant and placing it in a water bath on a nitrogen blower to concentrate, adding acetonitrile and mixing, and filtering to obtain the result.

[0017] Preferably, in step a, the preparation of described testing sample solution is that the root soil of aconite root, aconite root and aconite stem and leaf of different growth periods are dried, pulverized, crossed No. 3 sieve, accurately weighed 3g respectively and placed in 50mL polypropylene tool plug centrifuge tube, adding 15mL volume percent is 1% glacial acetic acid solution, vortex fully infiltrates the medicated powder, leaves standstill 30min, adds 15mL acetonitrile, vortex mixes, puts oscillator and shakes 5min, adds 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, shakes up, vibrates 3min, cools in ice bath for 10min, centrifuges 5 minutes, gets supernatant 9mL and is placed in the dispersed solid phase extraction cleanup tube that cleanup material is housed and makes purification completely, centrifuges 5 minutes, accurately draws supernatant 5mL and places nitrogen blow instrument and is concentrated to 4mL in 40 ℃ of water-baths, adds acetonitrile and is diluted to 1.0mL, mixes, and filters and gets final product.

[0018] Furthermore, in step a, the purification material is 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.

[0019] The beneficial effects of the present invention are:

[0020] The present invention adopts DESI (electrospray ionization imaging) to carry out mass spectrometry imaging to the paclobutrazol in the root soil of aconite root and / or the different parts of aconite root, compared with liquid chromatography commonly used in the prior art, the inventive method can be faster and more intuitive, reflects the distribution of the paclobutrazol in the different parts of aconite root and the content difference of the paclobutrazol in the different parts by imaging diagram, and can obtain more information than liquid chromatography. The inventive method can realize the real-time monitoring of its plant growth regulator by aconite root in the growth process, thus grasps the growth of aconite root. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The distribution of soil samples from the root of Aconite Root and samples from different parts of Aconite Root in Example 1.

[0022] Figure 2 These are the paclobutrazol mass spectrometry images of the soil from the roots of aconite root and samples from different parts of aconite root in Example 1.

[0023] Figure 3 To verify the response value of paclobutrazol in the soil around the roots of aconite and samples from different parts of aconite in Example 1. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below through specific examples.

[0025] Example 1

[0026] a. Preparation of test sample and reference solution

[0027] Gather the aconite root growth soil, the aconite root aerial part (stem and leaf), the aconite root underground part (root) at different time points according to Table 1, store as for-80 ℃ after gathering completes, take out during detection, dry respectively, pulverize, cross No. 3 sieve, accurately weighed powder 3g respectively places 50mL polystyrene tool plug centrifuge tube, add 15mL1% glacial acetic acid solution, vortex fully infiltrates the medicated powder, places 30min, add acetonitrile 15mL, vortex mixing, place vigorous vibration (500 times / minute) 5min on the oscillator, add the mixed powder of 6g anhydrous magnesium sulfate and 1.5g anhydrous sodium acetate, shake up immediately, place vigorous vibration (500 times / minute) 3min on the oscillator again, in Cool in an ice bath for 10 minutes, centrifuge (4000 revolutions per minute) for 5 minutes, take 9 mL of the supernatant, place it in a dispersed solid phase extraction purification tube pre-loaded with purification materials, 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, 90 mg of graphitized carbon black, vortex to mix thoroughly, place it on an oscillator and vigorously oscillate (500 times / min) for 5 minutes to completely purify it, centrifuge (4000 revolutions per minute) for 5 minutes, accurately draw 5 mL of the supernatant, place it on a nitrogen blower in a 40°C water bath and concentrate it to 0.4 mL, add acetonitrile to dilute it to 1.0 mL, vortex to mix, filter, and take the filtrate to obtain.

[0028] Accurately weigh an appropriate amount of paclobutrazol reference substance, dissolve it in methanol, and prepare a solution containing 1-1000 μg of paclobutrazol per 1 mL.

[0029] b. Sample

[0030] Punch a hole with a diameter of 4 mm on the filter paper, take 2 μL of the sample solution and the reference solution respectively for spotting, dry them, and place them on a conductive glass plate. For specific spotting sample information, see Figure 1 .

[0031] c. Detection

[0032] A conductive glass plate was placed on a mass spectrometer for examination. A positive ion full scan using a DESI (desorption electrospray ionization) source was used with a spray voltage of 3.5 kV, a scan range of 100-600 m / z, a spatial resolution of 110 μm, a nebulizer pressure of 0.6 MPa, an aqueous solution containing 0.4 wt% formic acid and 98 wt% methanol, and an ion source temperature of 160°C. Based on the imaging results, the residual paclobutrazol in the sample was determined.

[0033] The obtained raw data were analyzed by HDI software. The results are as follows: Figure 2 As shown, Figure 1Correspondingly, the yellow ones are the paclobutrazol reference substances, the light blue ones are mainly samples of collected soil, and the other black samples indicate that paclobutrazol was not detected.

[0034] Table 1 Sample source information

[0035]

[0036]

[0037] Verification Example 1

[0038] The liquid chromatography-mass spectrometry method is used to detect paclobutrazol in the soil and different parts of aconite root, including the following steps:

[0039] A. Prepare the test sample and reference solution as in step a of Example 1;

[0040] B. Instrumental analysis conditions; Chromatographic conditions: Chromatographic column: Acquity UPLC R BEH C18 column (2.1 mm × 100 mm, 1.7 μm); mobile phase: 0.1% ammonium formate in water (A)-0.1% ammonium formate in acetonitrile (B); gradient elution program as shown in Table 2 below; flow rate 0.4 mL min -1 , column temperature 40℃, injection volume 1μL.

[0041] Table 2 UPLC gradient elution program

[0042] Time (t / min) 0.1% ammonium formate aqueous solution (A) 0.1% ammonium formate in acetonitrile (B) 0 95 5 3 70 30 5 30 70 6 0 100 11 0 100 12 95 5 14 95 5

[0043] Q-TOF / MS conditions

[0044] Mass spectrometry was performed on a Waters SYNAPT G2 HDMS system. Nitrogen was used as the nebulizer and cone gas, source temperature: 150°C, reverse cone gas flow: 50 L / h, desolvation temperature: 450°C, desolvation gas flow: 800 L / h, sampling cone voltage: 40 V, extraction cone voltage: 4 V, capillary voltage: 2.5 kV in positive ion mode, scan time: 0.2 s, interscan time: 0.02 s, mass-to-charge ratio: m / z 100-600 Da, and lock mass: leucine enkephalin: m / z 556.2766 in positive ion mode (ESI+).

[0045] C. Data analysis.

[0046] The collected samples were collected by liquid chromatography-mass spectrometry data acquisition and analysis, and it was found that the soil samples contained a certain amount of paclobutrazol, and the content of paclobutrazol was higher in some samples above and below the ground. Figure 3 , the response values ​​of each sample in the figure basically match the response values ​​of the above-mentioned mass spectrometry imaging.

[0047] As can be seen from the above examples, the method of the present invention combines mass spectrometry imaging with liquid chromatography-mass spectrometry for verification, and the test results are basically consistent. The method of the present invention can achieve high-resolution mass spectrometry imaging of paclobutrazol in aconite planting soil and different parts of aconite, thereby enabling rapid and intuitive analysis of paclobutrazol residues in samples.

Claims

1. The method for detecting paclobutrazol in aconite soil and / or aconite different positions is characterized in that: The steps include: A, prepare testing sample solution and reference substance solution; The preparation of described testing sample solution is respectively with the root soil of aconite root, aconite root and aconite stem and leaf of different growth period, pulverize, sieve, add glacial acetic acid solution and leave standstill, add acetonitrile to mix, add mixed powder of anhydrous magnesium sulfate and anhydrous sodium acetate, vibrate, cool, centrifuge, take supernatant and be placed in the dispersed solid phase extraction purification tube that purification material is housed and make purification completely, centrifuge, take supernatant and be placed in nitrogen purge instrument water bath and concentrate, add acetonitrile to mix, filter and obtain; b. Punch a hole on the filter paper, draw the sample solution and the reference solution respectively, dry them, and place them on a conductive glass plate; c. DESI is used to detect the spotted sample, and mass spectrometry imaging scanning is performed. Based on the imaging results, the residual paclobutrazol in the sample to be tested is determined; the conditions of the DESI detection are: positive ion full scan, spray voltage 3.5-3.9 kV; scanning range: 100-600 m / z; spatial resolution 110-200 μm; sprayer pressure 0.6-0.8 MPa; ion source temperature 160-180° C.; the atomizer used in the DESI detection is an aqueous solution containing 0.35-0.45 wt% formic acid and 98-100 wt% methanol.

2. the method for detecting paclobutrazol in aconite soil and / or aconite different positions according to claim 1, is characterized in that: In step b, the amount of the sample solution to be tested is 2 to 10 μL.

3. the method for paclobutrazol in detection aconite root soil and / or aconite root different positions according to claim 1 and 2, is characterized in that: In step b, the diameter of the holes punched on the filter paper is 4 to 10 mm.

4. the method for detecting paclobutrazol in aconite soil and / or aconite different positions according to claim 1, is characterized in that: In step c, the DESI detection conditions are: positive ion full scan, spray voltage 3.5 kV; scan range: 100-600 m / z; spatial resolution 110 μm; sprayer pressure 0.6 MPa; and ion source temperature 160° C.

5. the method for detecting paclobutrazol in aconite soil and / or aconite different positions according to claim 1, is characterized in that: In step c, the atomizer used in the DESI detection is an aqueous solution containing 0.4 wt % formic acid and 98 wt % methanol.

6. the method for detecting paclobutrazol in aconite soil and / or aconite different positions according to claim 1, is characterized in that: Among the step a, the preparation of described testing sample solution is that the root soil of aconite root, aconite root and aconite stem and leaf of different growth periods are dried, pulverized, cross No. 3 sieve, accurately weigh 3g respectively and be placed in 50mL polypropylene tool plug centrifuge tube, adding 15mL volume percent is 1% glacial acetic acid solution, vortex fully infiltrates medicated powder, leaves standstill 30min, adds 15mL acetonitrile, vortex mixes, puts oscillator concussion 5min, adds 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, shakes up, vibrates 3min, cools off 10min in ice bath, centrifugal 5 minutes, gets supernatant 9mL and is placed in the dispersed solid phase extraction cleanup tube that cleanup material is housed and makes purification completely, centrifugal 5 minutes, accurately draw supernatant 5mL and be placed in nitrogen blow instrument and be concentrated to 4mL in 40 ℃ of water-baths, add acetonitrile and be diluted to 1.0mL, mix, filter and get final product.

7. the method for paclobutrazol in detection aconite root soil and / or aconite root different positions according to claim 1 or 6, is characterized in that: In step a, the purification material is 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.

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