Method for testing residual quantity of paclobutrazol and abscisic acid in plant growth regulator

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a method for testing residual quantity of paclobutrazol and abscisic acid in a plant growth regulator. According to the test method provided by the invention, sample pretreatment and detection conditions are optimized, and acetonitrile extraction, HLB solid phase extraction column purification and high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) are adopted for detection. The detection limit is as low as 2 [mu] g / L, the quantification limit is 10 [mu] g / L, the recovery rate range is 80.16%-109.60%, and the repeatability RSD is less than or equal to 4.1%. The method provided by the invention solves the problems of low sensitivity, complex operation, poor matrix interference resistance and the like in the prior art, has the advantages of high efficiency, sensitivity and reliability, and is suitable for detecting the residual plant growth regulator in complex matrixes such as traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and particularly relates to a method for testing the residues of paclobutrazol and abscisic acid in plant growth regulators. Background Art

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

[0003] Currently, the detection techniques for paclobutrazol and abscisic acid mainly include gas chromatography (GC), high-performance liquid chromatography (HPLC), and their combination with gas chromatography-mass spectrometry (GC-MS). However, the existing methods have significant limitations in terms of sensitivity, operation complexity, and anti-matrix interference ability. For example, Chinese Patent Application CN104897805A discloses a method for rapidly detecting the residue of paclobutrazol in traditional Chinese medicinal materials of plants, which mainly conducts content analysis based on gas chromatography. However, this method requires multiple solvent extractions, column chromatography purification, and multiple concentration steps. The operation steps are cumbersome, and the column chromatography parameters need to be precisely controlled. In addition, due to the interference of complex components such as polysaccharides and pigments in traditional Chinese medicinal materials on chromatographic separation, and the lack of qualitative ability of GC-ECD (gas chromatography-electron capture detector) in mass spectrometry, relying solely on retention time for judgment is prone to false positives, so it may have a certain impact on the accuracy of the test.

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

[0005] In order to solve the above technical problems, the present invention provides a method for testing the residues of paclobutrazol and abscisic acid in plant growth regulators, including the following steps:

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

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

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

[0009] S4. Purify the crude product solution using a solid-phase extraction column, collect all the purified solution, and use high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) to test the residues of paclobutrazol and abscisic acid.

[0010] As an implementable case, the mesh number of the ground and sieved sample is 50 - 100 mesh.

[0011] Further, the mesh number of the ground and sieved sample is 50 mesh.

[0012] Sieving and grinding treatment with a 50-mesh sieve can ensure that the particle size of the sample powder to be tested is uniform, reduce the fluctuation of extraction efficiency caused by particle size differences, and improve the experimental repeatability and data comparability; in addition, due to the sealed design of the stoppered centrifuge tube, it can prevent the volatilization of the organic phase solvent after use. As a starting point for standardized sample pretreatment, it can avoid the loss of the target substances paclobutrazol and abscisic acid, which affects the accuracy and precision of the test.

[0013] As an implementable case, the inorganic salts include one of sodium chloride, potassium chloride, sodium sulfate, magnesium sulfate, or ammonium sulfate.

[0014] Further, the inorganic salts include sodium chloride.

[0015] Sodium chloride increases the ionic strength in the solution, reduces the solubility of the target substances paclobutrazol and abscisic acid in the aqueous phase, promotes their distribution into the organic phase, and improves the extraction efficiency; at the same time, the shaking operation enables sodium chloride to fully contact the sample powder to be tested, prevents caking, ensures uniform salting-out effect, and optimizes the release and distribution of the target substances from the complex matrix.

[0016] As an implementable case, the organic phase includes acetonitrile.

[0017] As an implementable case, the rotation speed of the homogenization treatment is greater than 12000 rpm.

[0018] Further, the rotation speed of the homogenization treatment is 16000 rpm.

[0019] As an implementable case, the rotational speed of the centrifugation is 3000 - 6000 rpm.

[0020] Furthermore, the rotational speed of the centrifugation 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. At the same time, it can disrupt the plant cell structure, release intracellular components, completely break the cell wall through high-shear homogenization treatment, increase the contact area between the solvent and the target substance, and shorten 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 blockage of the subsequent purification column by solid particles.

[0022] As an implementable case, the packing material of the solid-phase extraction column is a hydrophilic-lipophilic balance packing material.

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

[0024] The hydrophilic-lipophilic balance packing material can simultaneously adsorb polar and non-polar compounds, effectively remove matrix interferences such as pigments, polysaccharides, and lipids in the analyte, significantly reduce miscellaneous interferences, and improve the signal-to-noise ratio and accuracy of LC-MS / MS detection.

[0025] As an implementable case, in the LC-MS / MS detection, the specifications of the chromatographic column are 2.1×100 mm×1.8 μm, and the model is Wasters HSS T3.

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

[0027] Column temperature: 30 - 40 °C;

[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 min.

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

[0032] Column temperature: 35 °C;

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

[0034] Mobile phase B: A mixed solvent of mobile phase A and acetonitrile with a volume ratio of 5:95.

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

[0036] Table 1

[0037] Serial number Detected substance Q1 Q3 DP(V) CE(V) 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] (1) The test method provided by the present invention mainly combines LC-MS / MS with the multiple reaction monitoring mode. The detection limits of paclobutrazol and abscisic acid are both as low as 2 μg / L, and the quantification limit is 10 μg / L, which is significantly better than the traditional GC or HPLC-UV methods and can meet the strict detection requirements for trace residues.

[0040] (2) The detection method provided by the present invention can simultaneously detect two plant growth regulators, paclobutrazol and abscisic acid. By optimizing the mass spectrometry parameters, multi-ion pair monitoring is achieved, reducing the cost and time of repeated detection.

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

[0042] (4) The test method provided by the present invention has a wide recovery range. The recovery rate of paclobutrazol is 93.72% - 97.56%, and the recovery rate of abscisic acid is 80.16% - 109.60%, which meets the pharmacopoeia standards; and the repeatability RSD ≤ 4.1% and precision RSD ≤ 15% of the detection method provided by the present invention are significantly better than the traditional methods.

[0043] (5) Verified by the durability test, the test method provided by the present invention uses different equipment for actual operation analysis, and the detection results have high consistency. The chromatographic conditions and mass spectrometry parameters can be slightly adjusted without affecting the results, which is applicable to different laboratory environments and meets the standardized requirements of General Chapters 9101 and 9099 of the Chinese Pharmacopoeia. Description of the drawings

[0044] Figure 1 It is the TIC ion chromatogram of the durability test in Example 1. Detailed implementation manners

[0045] Example 1

[0046] This example provides a test method for the residues of paclobutrazol and abscisic acid in plant growth regulators, including the following steps:

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

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

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

[0050] S4. Purify the crude product solution using an HLB SPE solid-phase extraction column, collect all the purified solution, and use the LC-MS / MS method to test the residues of paclobutrazol and abscisic acid.

[0051] In the LC-MS / MS described above, the specifications of the liquid chromatography column are 2.1×100 mm×1.8 μm, and the model is WastersHSS T3

[0052] The conditions of the liquid phase are as follows:

[0053] Column temperature: 35 °C;

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

[0055] Mobile phase B: A mixed solvent of mobile phase A and acetonitrile with a volume ratio of 5:95;

[0056] The gradient elution conditions of the liquid phase are shown in Table 2;

[0057] Table 2

[0058]

[0059] The mass spectrometry conditions of the LC-MS / MS described above are shown in Table 3;

[0060] Table 3

[0061] Serial number Detected substance Q1 Q3 DP(V) CE(V) 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. Linear relationship test

[0063] Construct a linear equation, where the injection concentrations are set as: 2 μg / L, 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, test the response values of each concentration, and the experimental test results are shown in Table 4 for details.

[0064] Table 4

[0065]

[0066]

[0067] In this experiment, different concentrations were injected, and the linear R was all > 0.99, indicating that the linear relationship between concentration and response value was significant, stable within the tested concentration range, with good repeatability, and it could be used for accurate quantitative analysis of the residues of abscisic acid and paclobutrazol in unknown samples.

[0068] 2. Detection limit test

[0069] Take 1.0 mL (10 components) of the purified solution respectively, place it on a nitrogen blowing instrument, concentrate it to a volume of about 0.4 mL in a 40 °C water bath, add 2 μL of the reference substance (standard mixed solution of paclobutrazol and abscisic acid, with a concentration of 1 μg / mL) solution, then dilute it to 1 mL with acetonitrile, vortex and mix well, and detect the signal-to-noise ratio of each component. The experimental results are shown in Tables 5 - 6.

[0070] Table 5

[0071]

[0072] Table 6

[0073]

[0074]

[0075] It can be seen from the experimental results in Tables 5 - 6 that in this experiment, 2 μL of the mixed reference substance solution was added to the blank matrix (blank matrix sample that was detected to contain no analytes, i.e., residues of paclobutrazol and abscisic acid), diluted to 1 mL with acetonitrile, and measured in parallel 10 times. The signal-to-noise ratio was all > 3, indicating that this method could reliably detect the target compounds paclobutrazol and abscisic acid, and had good anti-interference ability and reproducibility, both meeting the requirements of the pharmacopoeia.

[0076] 3. Quantitation limit detection

[0077] Take 1.0 mL (10 components) of the purified solution respectively, place it on a nitrogen blowing instrument, concentrate it to a volume of about 0.6 mL in a 40 °C water bath, add 10 μL of the reference substance (standard mixed solution of paclobutrazol and abscisic acid, with a concentration of 1 μg / mL) solution, then dilute it to 1 mL with acetonitrile, vortex and mix well, and detect the signal-to-noise ratio of each component. The experimental results are shown in Tables 7 - 8.

[0078] Table 7

[0079]

[0080] Table 8

[0081]

[0082]

[0083] In this experiment, 10 μL of the mixed reference solution was added to the blank matrix (a blank matrix sample that was tested and found to contain no residues of the analytes, i.e., paclobutrazol and abscisic acid), and the determination was carried out in parallel 10 times. The signal-to-noise ratio was >10 for all measurements, indicating that this method can accurately quantify the target compounds, and the precision, anti-interference ability, and robustness all meet the requirements of the pharmacopoeia.

[0084] 4. Precision test

[0085] Take 1.0 mL (6 components) of the purified solution respectively, place it on a nitrogen evaporator, concentrate it to a volume of about 0.4 mL in a 40 °C water bath, add 10 μL of the reference solution (a standard mixed solution of paclobutrazol and abscisic acid with a concentration of 1 μg / mL), then dilute it to 1 mL with acetonitrile, vortex and mix well, and detect the determined concentration of the spiked samples of each component. The experimental results are shown in Table 9.

[0086] Table 9

[0087]

[0088] In this experiment, 10 μL of the mixed reference solution was added to the blank matrix, and the determination was carried out in parallel 6 times. The relative standard deviation of repeatability was ≤15% for all measurements.

[0089] 5. Quantitative determination

[0090] Take the purified solution, add the reference solution, vortex and mix well, and detect the maximum recovery rate and minimum recovery rate of the spiked samples of each component. The experimental results are shown in Table 10.

[0091] Table 10

[0092]

[0093] In this experiment, 100 μL of the mixed reference solution 2 (a mixed standard solution of paclobutrazol and abscisic acid with a concentration of 0.1 μg / mL) was added during the pretreatment process of the blank matrix, and the determination was carried out in parallel 6 times. The recovery rate range of the test samples was 80.16 - 109.60%, and the standard recovery rate range was 60.0 - 130.0%.

[0094] 6. Robustness test

[0095] Robustness refers to the degree of tolerance of the determination results to be unaffected when there are minor changes in the determination conditions. In this experiment, the robustness test was carried out using the same chromatographic column and mass spectrometry conditions on different equipment, and the TIC ion current diagram is as Figure 1 shown, and the specific parameters are shown in Table 11.

[0096] Table 11

[0097] Detected substance Quantitative ion pair Retention time Scan mode Collision voltage (V) Paclobutrazol 294.2-＞70.1 5.129 Positive 20 Abscisic acid 262.8-＞153.1 6.725 Negative 10

[0098] The durability experiment shows that different devices are used in this experimental test method for the durability experiment, and the results meet the durability requirements.

Claims

1. A method for testing the residual amount of paclobutrazol and abscisic acid in a plant growth regulator, characterized in that: The following steps are involved: S1. Grind and sieve the sample to be tested, place it in a stoppered centrifuge tube, add inorganic salt, shake it evenly, add the organic phase, homogenize it for 1-3 minutes, then centrifuge it, let it stand, and collect the precipitate and the first supernatant; S2, add the organic phase to the precipitate, homogenize for 1-3 minutes, centrifuge, and collect the second supernatant; S3, combining the first supernatant and the second supernatant, concentrating under reduced pressure to 5-10 mL, then cooling to 20-30° C., adding the organic phase to dilute, shaking well, and obtaining a crude product solution; S4. The crude product solution was purified using a solid phase extraction column, the purified solution was collected, and the residual amounts of paclobutrazol and abscisic acid were tested using LC-MS / MS.

2. The method for testing the residual amounts of paclobutrazol and abscisic acid in a plant growth regulator according to claim 1, characterized in that: The mesh number of the grinding and sieving sieve is 50-100 meshes.

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 organic phase comprises acetonitrile.

4. The method for testing the residual amounts of paclobutrazol and abscisic acid in a plant growth regulator according to claim 1, characterized in that: The inorganic salt includes one of sodium chloride, potassium chloride, sodium sulfate, magnesium sulfate or ammonium sulfate.

5. The method for testing the residual amounts of paclobutrazol and abscisic acid in a plant growth regulator according to claim 1, characterized in that: The rotation speed of the homogenization treatment is greater than 12000 rpm.

6. The method for testing the residual amounts of paclobutrazol and abscisic acid in a plant growth regulator according to claim 1, characterized in that: The centrifugal rotation speed is 3000-6000rpm.

7. The method for testing the residual amounts of paclobutrazol and abscisic acid in a plant growth regulator according to claim 1, characterized in that: The filler of the solid phase extraction column is a hydrophilic-lipophilic balanced filler.

8. The method for testing the residual amounts of paclobutrazol and abscisic acid in a plant growth regulator according to claim 7, characterized in that: The solid phase extraction column is an HLB SPE solid phase extraction column.

9. The method for testing the residual amount of paclobutrazol and abscisic acid in a plant growth regulator according to any one of claims 1 to 8, characterized in that: In the LC-MS / MS detection, the chromatographic column has a specification of 2.1×100 mm×1.8 μm and a model of Wasters HSS T3.

10. The method for testing the residual amounts of paclobutrazol and abscisic acid in plant growth regulators according to claim 9, characterized in that: In the LC-MS / MS detection, the liquid phase conditions are: Column temperature is 30-40°C; Mobile phase A was ammonium formate containing formic acid; Mobile phase B is a mixed solvent of mobile phase A and acetonitrile; The running time is 10-20 minutes.

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