Method for testing residual amounts of paclobutrazol and abscisic acid in plant growth regulators

Abstract

The present application relates to the technical field of analytical chemistry, in particular to a kind of test method for residual amount of paclobutrazol and abscisic acid in plant growth regulator.The test method provided by the present application extracts by acetonitrile, purifies by HLB solid-phase extraction column and detects by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) by optimizing sample pretreatment and detection conditions.The detection limit is as low as 2 μg / L, the quantification limit is 10 μg / L, the recovery rate ranges from 80.16% to 109.60%, and the repeatability RSD is less than or equal to 4.1%.The present application solves the problems of low sensitivity, complex operation and poor anti-matrix interference ability in the prior art, has the advantages of high efficiency, sensitivity and reliability, and is suitable for the detection of plant growth regulator residues in complex matrix such as traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators. Background Technology

[0002] Plant growth regulators (PGRs) are indispensable chemical substances in modern agriculture. After application, they can significantly improve crop yield and quality by regulating plant physiological processes. Paclobutrazol and abscisic acid (ABA) are two important classes of PGRs: Paclobutrazol slows plant growth by inhibiting gibberellin synthesis and is widely used for dwarfing regulation in fruit trees and flowers; ABA mainly participates in plant stress responses, enhancing crop resistance under drought conditions. However, excessive use or improper residues can accumulate through the food chain, posing potential threats to the ecological environment and human health, such as endocrine disruption, reproductive toxicity, and the risk of chronic diseases. Therefore, establishing efficient, sensitive, and reliable methods for detecting PGR residues is of great significance for ensuring agricultural product safety and environmental sustainability.

[0003] Currently, the main detection techniques for paclobutrazol and abscisic acid include gas chromatography (GC), high-performance liquid chromatography (HPLC), and their combined GC-MS. However, existing methods have significant limitations in terms of sensitivity, operational complexity, and resistance to matrix interference. For example, Chinese invention patent application CN104897805A discloses a method for rapid detection of paclobutrazol residues in medicinal plants, which is mainly based on gas chromatography for content analysis. However, this method requires multiple solvent extractions, column purification, and multi-step concentration, making the operation cumbersome and requiring precise control of column parameters. In addition, complex components such as polysaccharides and pigments in medicinal plants may interfere with chromatographic separation, and GC-ECD (gas chromatography-electron capture detector) lacks the qualitative capabilities of mass spectrometry, relying solely on retention time for judgment, which can easily lead to false positives. Therefore, it may have a certain impact on the accuracy of the test.

[0004] Therefore, there is an urgent need for a new testing method for the residual amounts of paclobutrazol and abscisic acid in plant growth regulators, so as to better detect the content of paclobutrazol or abscisic acid in Chinese medicinal materials such as licorice. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators, comprising the following steps:

[0006] S1. Grind and sieve the sample to be tested, place it in a 100mL stoppered centrifuge tube, add inorganic salt, shake well, add organic phase, homogenize for 1-3 minutes, then centrifuge, let stand, and collect the precipitate and the first supernatant.

[0007] S2. Add organic phase to precipitate, homogenize for 1-3 min, centrifuge, and collect second supernatant;

[0008] S3. Combine the first and second supernatants, concentrate under reduced pressure to 5-10 mL, then cool to 20-30℃, add organic phase to dilute, shake well, and obtain crude product solution;

[0009] S4. The crude solution was purified using a solid-phase extraction column. All purified liquid was collected and the residual amounts of paclobutrazol and abscisic acid were tested using high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0010] As an example of implementation, the grinding and sieving process is described as having a mesh size of 50-100.

[0011] Furthermore, the mesh size of the grinding and sieving process is 50 mesh.

[0012] Sieving and grinding through a 50-mesh sieve ensures uniform particle size of the sample powder, reduces fluctuations in extraction efficiency due to differences in particle size, and improves experimental repeatability and data comparability. In addition, the stoppered centrifuge tubes, due to their sealed design, prevent the evaporation of organic solvents after use. As a starting point for standardized sample pretreatment, this avoids the loss of target substances such as paclobutrazol and abscisic acid, which could affect the accuracy and precision of the test.

[0013] As an example of implementation, the inorganic salt includes one of sodium chloride, potassium chloride, sodium sulfate, magnesium sulfate, or ammonium sulfate.

[0014] Furthermore, the inorganic salt includes sodium chloride.

[0015] Sodium chloride increases the ionic strength in the solution, reduces the solubility of the target compounds paclobutrazol and abscisic acid in the aqueous phase, promotes their distribution to the organic phase, and improves the extraction efficiency. At the same time, the shaking operation ensures that sodium chloride is in full contact with the sample powder, prevents agglomeration, ensures uniform salting out, and optimizes the release and distribution of the target compounds from the complex matrix.

[0016] As an example of an implementable approach, the organic phase includes acetonitrile.

[0017] As an example of implementation, the homogenization process is performed at a speed greater than 12,000 rpm.

[0018] Furthermore, the rotation speed of the homogenization process is 16,000 rpm.

[0019] As an example of implementation, the centrifugation speed is 3000-6000 rpm.

[0020] Furthermore, the centrifugation speed is 4000 rpm.

[0021] Acetonitrile is a polar organic solvent with both hydrophilic and lipophilic properties. It can effectively dissolve target substances such as paclobutrazol and abscisic acid, while destroying plant cell structure and releasing intracellular components. High-shear homogenization thoroughly breaks down cell walls, increasing the contact area between the solvent and the target substance and shortening the extraction time. Centrifugation can separate the solid precipitate from the supernatant containing the target substance, reduce matrix interference, collect the organic phase enriched with the target substance, and avoid solid particles clogging the subsequent purification column.

[0022] As an example of implementation, the solid-phase extraction column is packed with a hydrophilic-lipophilic balanced packing agent.

[0023] Furthermore, the solid phase extraction column is an HLB SPE solid phase extraction column.

[0024] Hydrophilic and lipophilic balanced fillers can simultaneously adsorb polar and nonpolar compounds, effectively removing matrix interferences such as pigments, polysaccharides, and lipids from the analyte, significantly reducing extraneous interferences, and improving the signal-to-noise ratio and accuracy of LC-MS / MS detection.

[0025] As an example of feasible implementation, the LC-MS / MS detection uses a chromatographic column with dimensions of 2.1×100mm×1.8μm and a Wasters HSS T3 model.

[0026] Furthermore, in the LC-MS / MS detection, the liquid phase conditions are as follows:

[0027] Column temperature: 30-40℃;

[0028] Mobile phase A: Ammonium formate containing formic acid;

[0029] Mobile phase B: A mixed solvent of mobile phase A and acetonitrile;

[0030] Running time: 10-20 minutes.

[0031] Furthermore, the conditions for the liquid phase are as follows:

[0032] Column temperature: 35℃;

[0033] Mobile phase A: 10 mmol / L ammonium formate solvent with a formic acid mass concentration of 0.05 wt%;

[0034] Mobile phase B: A mixture of mobile phase A and acetonitrile in a volume ratio of 5:95.

[0035] Furthermore, the mass spectrometry conditions for the LC-MS / MS are shown in Table 1;

[0036] Table 1

[0037] 1 Paclobutrazol 1 294.2 70.1 85 60 2 Paclobutrazol 2 294.2 125.1 85 50 3 Paclobutrazol 3 294.2 165.1 85 35 4 abscisic acid 1 263.1 153.1 -100 -25 5 abscisic acid 2 263.1 204.1 -100 -25 6 abscisic acid 3 263.1 219.1 -100 -20

[0038] Beneficial effects

[0039] (i) The test method provided by this invention mainly uses LC-MS / MS combined with multiple reaction monitoring mode. The detection limits of paclobutrazol and abscisic acid are as low as 2 μg / L and the quantitation limit is 10 μg / L, which is significantly better than the traditional GC or HPLC-UV methods and can meet the strict detection requirements of trace residues.

[0040] (ii) The detection method provided by the present invention can simultaneously detect two plant growth regulators, paclobutrazol and abscisic acid. By optimizing mass spectrometry parameters, multi-ion pair monitoring can be achieved, reducing the cost and time of repeated detection.

[0041] (III) This invention mainly uses acetonitrile extraction combined with HLB SPE solid phase extraction column purification to effectively remove complex matrix components such as polysaccharides and pigments from licorice medicinal materials. The signal-to-noise ratio is >3, which can ensure the reliability of the detection results.

[0042] (iv) The test method provided by the present invention has a wide range of recovery rates, with the recovery rate of paclobutrazol being 93.72%-97.56% and the recovery rate of abscisic acid being 80.16%-109.60%, which meets the pharmacopoeia standards; and the detection method provided by the present invention has a repeatability RSD≤4.1% and a precision RSD≤15%, which is significantly better than traditional methods.

[0043] (v) Through durability testing, the test method provided by this invention has been verified to be highly consistent with the results when used in actual analysis with different equipment. The chromatographic conditions and mass spectrometry parameters can be finely adjusted without affecting the results. It is suitable for different laboratory environments and meets the standardization requirements of General Chapters 9101 and 9099 of the Chinese Pharmacopoeia. Attached Figure Description

[0044] Figure 1 This is a TIC ion flow map from the durability test in Example 1. Detailed Implementation

[0045] Example 1

[0046] This embodiment provides a method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators, including the following steps:

[0047] S1. Grind the licorice medicinal material sample to be tested, sieve it through a 50-mesh sieve, place it in a 100mL stoppered centrifuge tube, add 1g of sodium chloride, shake it evenly, add 50mL of acetonitrile, homogenize it at 16000rpm for 1min, then centrifuge it at 4000rpm, let it stand, and collect the precipitate and the first supernatant.

[0048] S2. Add 50 mL of acetonitrile to the precipitate, homogenize at 16000 rpm for 1 min, centrifuge at 4000 rpm, and collect the second supernatant.

[0049] S3. Combine the first and second supernatants, concentrate under reduced pressure to 5-6 mL, then cool to 25°C, add acetonitrile to dilute to 10 mL, shake well to obtain the crude product solution;

[0050] S4. The crude solution was purified using an HLB SPE solid-phase extraction column. All purified solution was collected and the residual amounts of paclobutrazol and abscisic acid were tested using LC-MS / MS.

[0051] In the LC-MS / MS described above, the liquid chromatography column has dimensions of 2.1 × 100 mm × 1.8 μm and is a Wasters HSS T3 model.

[0052] The conditions for the liquid phase are:

[0053] Column temperature: 35℃;

[0054] Mobile phase A: 10 mmol / L ammonium formate solvent with a formic acid mass concentration of 0.05 wt%;

[0055] Mobile phase B: a mixture of mobile phase A and acetonitrile in a volume ratio of 5:95;

[0056] The gradient elution conditions for the liquid phase are shown in Table 2.

[0057] Table 2

[0058]

[0059] The mass spectrometry conditions for the LC-MS / MS are shown in Table 3.

[0060] Table 3

[0061] 1 Paclobutrazol 1 294.2 70.1 85 60 2 Paclobutrazol 2 294.2 125.1 85 50 3 Paclobutrazol 3 294.2 165.1 85 35 4 abscisic acid 1 263.1 153.1 -100 -25 5 abscisic acid 2 263.1 204.1 -100 -25 6 abscisic acid 3 263.1 219.1 -100 -20

[0062] 1. Linearity test

[0063] A linear equation was constructed with injection concentrations of 2 μg / L, 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L, and 100 μg / L. The response values ​​for each concentration were tested, and the experimental results are detailed in Table 4.

[0064] Table 4

[0065]

[0066]

[0067] This experiment used different concentrations for injection, and the linearity R was >0.99 for all cases, indicating a significant linear relationship between concentration and response value. The results showed stability and good repeatability within the test concentration range, and the sample can be used for accurate quantitative analysis of abscisic acid and paclobutrazol residues in unknown samples.

[0068] 2. Detection limit test

[0069] Take 1.0 mL of the purified solution (10 components) and place it on a nitrogen blower. Concentrate it to a volume of about 0.4 mL in a 40 °C water bath. Add 2 μL of the reference solution (a mixed solution of paclobutrazol and abscisic acid, with a concentration of 1 μg / mL). Dilute with acetonitrile to 1 mL, vortex to mix, and detect the signal-to-noise ratio of each component. The experimental results are detailed in Tables 5-6.

[0070] Table 5

[0071]

[0072] Table 6

[0073]

[0074]

[0075] As can be seen from the experimental results in Table 5-6, in this experiment, 2 μL of mixed reference solution was added to the blank matrix (a blank matrix sample that was tested and found to be free of the analytes, namely paclobutrazol and abscisic acid residues), and acetonitrile was added to make up to 1 mL. The results were repeated 10 times in parallel, and the signal-to-noise ratio was >3 in all cases. This indicates that the method can reliably detect the target compounds paclobutrazol and abscisic acid, and has good anti-interference ability and reproducibility, which meet the pharmacopoeia requirements.

[0076] 3. Limit of Quantitation Detection

[0077] Take 1.0 mL of the purified solution (10 components) and place it on a nitrogen blower. Concentrate it to a volume of about 0.6 mL in a 40 °C water bath. Add 10 μL of the reference solution (a mixed solution of paclobutrazol and abscisic acid, with a concentration of 1 μg / mL). Dilute with acetonitrile to 1 mL, vortex to mix, and detect the signal-to-noise ratio of each component. The experimental results are detailed in Tables 7-8.

[0078] Table 7

[0079]

[0080] Table 8

[0081]

[0082]

[0083] In this experiment, 10 μL of mixed reference solution was added to a blank matrix (a blank matrix sample that was tested to be free of the analytes, namely paclobutrazol and abscisic acid residues), and the results were measured in parallel 10 times. The signal-to-noise ratio was >10 in all cases, indicating that the method can accurately quantify the target compound, and that the precision, anti-interference ability and robustness meet the pharmacopoeia requirements.

[0084] 4. Precision testing

[0085] Take 1.0 mL of the purified solution (6 components) and place it on a nitrogen blower. Concentrate it to a volume of about 0.4 mL in a 40 °C water bath. Add 10 μL of the reference solution (a mixed solution of paclobutrazol and abscisic acid, with a concentration of 1 μg / mL). Then dilute it to 1 mL with acetonitrile. Vortex mix well and determine the concentration of each component by spiking. The experimental results are detailed in Table 9.

[0086] Table 9

[0087]

[0088] In this experiment, 10 μL of mixed standard solution was added to the blank matrix, and the results were measured in parallel 6 times. The relative standard deviation of the repeatability was ≤15% for all samples.

[0089] 5. Quantitative determination

[0090] Add the purified solution to the reference standard, vortex to mix, and detect the maximum and minimum recoveries of each component. The experimental results are detailed in Table 10.

[0091] Table 10

[0092]

[0093] In this experiment, 100 μL of mixed standard 2 (a mixed standard solution of paclobutrazol and abscisic acid, with a concentration of 0.1 μg / mL) was added during the blank matrix pretreatment process. The assay was performed in parallel for 6 times. The recovery rate of the test sample ranged from 80.16% to 109.60%, and the recovery rate of the standard ranged from 60.0% to 130.0%.

[0094] 6. Durability test

[0095] Robustness refers to the degree to which the measurement results remain unaffected by minor changes in measurement conditions. This experiment used different equipment with the same chromatographic column and mass spectrometry conditions to conduct robustness tests. The TIC ion chromatograms are shown below. Figure 1 As shown, the specific parameters are shown in Table 11.

[0096] Table 11

[0097] Paclobutrazol 294.2-＞70.1 5.129 Positive 20 abscisic acid 262.8-＞153.1 6.725 Negative 10

[0098] Durability tests show that the durability test method used in this experiment, employing different equipment, yields results that meet durability requirements.

Claims

1. A method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators, characterized in that, Includes the following steps: S1. Grind and sieve the sample to be tested, place it in a stoppered centrifuge tube, add inorganic salt, shake to mix evenly, add organic phase, homogenize for 1-3 minutes, then centrifuge, let stand, and collect the precipitate and the first supernatant. S2. Add organic phase to precipitate, homogenize for 1-3 min, centrifuge, and collect second supernatant; S3. Combine the first and second supernatants, concentrate under reduced pressure to 5-10 mL, then cool to 20-30℃, add organic phase to dilute, shake well, and obtain crude product solution; S4. The crude solution was purified using a solid-phase extraction column, the purified liquid was collected, and the residual amounts of paclobutrazol and abscisic acid were tested by LC-MS / MS. The organic phase includes acetonitrile; The inorganic salt mentioned is one of sodium chloride, potassium chloride, sodium sulfate, magnesium sulfate, or ammonium sulfate; The solid-phase extraction column is an HLB SPE solid-phase extraction column; In the LC-MS / MS detection described above, the chromatographic column specifications are 2.1×100mm×1.8μm, and the model is Wasters HSS T3; In the LC-MS / MS detection described above, the liquid phase conditions are as follows: The column temperature is 30-40℃; Mobile phase A is ammonium formate containing formic acid; Mobile phase B is a mixed solvent of mobile phase A and acetonitrile; Running time of mobile phase A: The mobile phase A is a 10 mmol / L ammonium formate solvent with a formic acid mass concentration of 0.05 wt% for 10-20 min; The mobile phase B is a mixed solvent of mobile phase A and acetonitrile in a volume ratio of 5:95; The gradient elution conditions for the liquid phase are shown in the table below: 。 2. The method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators according to claim 1, characterized in that, The mesh size of the sieve used for grinding and sieving is 50-100 mesh.

3. The method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators according to claim 1, characterized in that, The rotation speed of the homogenization process is greater than 12,000 rpm.

4. The method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators according to claim 1, characterized in that, The centrifuge speed is 3000-6000 rpm.

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