CN114264754A - Indolylacetic acid adsorbent and application thereof in indoleacetic acid detection - Google Patents
Indolylacetic acid adsorbent and application thereof in indoleacetic acid detection Download PDFInfo
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 title claims abstract description 235
- 239000003617 indole-3-acetic acid Substances 0.000 title claims abstract description 112
- 239000003463 adsorbent Substances 0.000 title claims abstract description 49
- 238000001514 detection method Methods 0.000 title claims abstract description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 153
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 27
- OKKRPWIIYQTPQF-UHFFFAOYSA-N Trimethylolpropane trimethacrylate Chemical compound CC(=C)C(=O)OCC(CC)(COC(=O)C(C)=C)COC(=O)C(C)=C OKKRPWIIYQTPQF-UHFFFAOYSA-N 0.000 claims abstract description 14
- XUGNJOCQALIQFG-UHFFFAOYSA-N 2-ethenylquinoline Chemical compound C1=CC=CC2=NC(C=C)=CC=C21 XUGNJOCQALIQFG-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000012719 thermal polymerization Methods 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 9
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Abstract
The invention relates to a preparation method of an indoleacetic acid adsorbent, which is characterized in that 2-vinyl quinoline, trimethylolpropane trimethacrylate and azodiphenyl are dissolved in methanol, sealed heating is carried out for thermal polymerization, then methanol and acetic acid are used for washing to obtain a finished product. The method for detecting the indoleacetic acid utilizes the synthesized novel adsorbent to adsorb and enrich the trace indoleacetic acid in the agricultural products, and adopts the high performance liquid chromatography to detect the content of the indoleacetic acid in the agricultural products, so that the extraction efficiency is high, the sensitivity is high, and the detection result is accurate.
Description
Technical Field
The invention relates to a method for detecting the content of indoleacetic acid in agricultural products by using a synthesized novel polymer adsorbent, belonging to the technical field of component detection.
Background
In recent years, a detection technology for auxin residues in fruits and vegetables attracts people's attention. Auxin (also known as indoleacetic acid, IAA) is an important plant hormone and influences the characteristics of cell division, growth, differentiation and the like in plants. Researches find that synthetic auxin such as indoleacetic acid plays an important role in plant growth regulation and control, and is widely applied to agricultural production. Thus, uncontrolled or inappropriate use can result in residues in the fruit or vegetable, which can adversely affect food safety and human health. The invention patent CN108303453A provides an IAA electrochemical method for detecting soybean seedlings, but the IAA electrochemical method is easily interfered by various backgrounds in plants, and the electrodes are made of expensive materials, so that the stability is insufficient, and the IAA detection of agricultural product residues is not involved. The literature also reports a method for measuring the content of indoleacetic acid in fermentation liquor by using a high performance liquid chromatography (tropical agricultural engineering, 2009, 05, 1-3). However, there is no effective way to enrich and detect trace IAA. Due to the complexity of the matrix and the trace amount of residual auxin in real food samples, there is a need for a reasonably efficient sample preparation method to clean it prior to instrumental analysis, these complex problems are not addressed in the above patents and literature, and it is therefore necessary to develop new methods to determine trace amounts of residual auxin in agricultural products.
To date, sample pre-treatment is the most critical and time-consuming step due to the low auxin concentration in plant tissues and the presence of interfering substances, requiring extensive purification prior to quantification. Such as dispersion microextraction and solid phase microextraction, have the advantages of strong enrichment capacity, low consumption of organic solvent, but poor recovery rate and reproducibility. And the fiber liquid micro-extraction needs less organic solvent and has strong enrichment capacity, but is limited to extracting ionizable compounds and the like from aqueous solution. The traditional liquid-liquid extraction method is one of the simplest methods for sample pretreatment, but needs the processes of crushing a large number of plants, homogenizing and the like, and causes serious loss of residual auxin.
Disclosure of Invention
The invention aims to provide an indoleacetic acid detection method with high extraction efficiency, high sensitivity and accurate detection result, an indoleacetic acid adsorbent used by the method and a preparation method of the indoleacetic acid adsorbent.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a preparation method of indoleacetic acid adsorbent comprises dissolving 2-vinylquinoline, trimethylolpropane trimethacrylate and azodiphenyl in methanol, sealing, heating, and performing thermal polymerization to obtain yellow powder; then, the powder is washed with methanol and acetic acid to remove unreacted monomers and byproducts in the synthesis process, and a finished product is obtained.
The molar ratio of the 4-2-vinylquinoline to the trimethylolpropane trimethacrylate to the azodiphenyl is (4-2) to (10-8) to (0.1-1). The temperature of the thermal polymerization reaction is 60-100 ℃, and the time of the thermal polymerization reaction is 10-30 h. The powder is washed by detergent prepared from methanol and acetic acid according to the volume ratio of 5: 1-100: 1.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: the indoleacetic acid adsorbent prepared by the preparation method is provided.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: an application of the indole acetic acid adsorbent in detection of residual indole acetic acid in agricultural products.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: an indoleacetic acid detection method comprises the following steps:
A. preparing an indoleacetic acid adsorbent: the 2-vinyl quinoline and trimethylolpropane trimethacrylate are subjected to cross-linking reaction to generate the indoleacetic acid adsorbent.
B. Preparing a sample solution to be tested: freezing, slicing, extracting and adsorbing the agricultural product, mixing the agricultural product with the indoleacetic acid adsorbent to ensure that the indoleacetic acid adsorbent absorbs and enriches indoleacetic acid, and treating the indoleacetic acid adsorbent with methanol to ensure that the indoleacetic acid adsorbent releases indoleacetic acid to obtain a sample solution to be detected;
C. determining the solution of the sample to be tested: taking the sample solution to be measured, and carrying out quantitative determination by a high performance liquid chromatograph.
The preparation method of the indoleacetic acid adsorbent in the step A comprises the following steps: dissolving 2-vinylquinoline, trimethylolpropane trimethacrylate and azodiphenyl in methanol, sealing and heating for thermal polymerization reaction to obtain yellow powder; then, the powder is washed with methanol and acetic acid to remove unreacted monomers and byproducts in the synthesis process, and a finished product is obtained.
The pretreatment of the agricultural product in the step B comprises the following steps: slicing and freezing an agricultural product, adding methanol into a freeze-dried agricultural product sample, stirring and centrifuging, taking supernate, transferring the supernate into a test tube, and adding the supernate into an indoleacetic acid adsorbent; stirring the obtained suspension at room temperature, centrifuging, and removing supernatant; adding methanol for washing for many times to remove the final interfering compound; then, treating the indoleacetic acid adsorbent with methanol with the concentration of 100% to release indoleacetic acid, stirring the obtained suspension at room temperature, centrifuging, and filtering supernatant to obtain a sample solution to be detected.
In the step C, the detection method of the high performance liquid chromatograph is 150mm multiplied by 2.1mm, a 3 μm Thermo Acclaim C30 chromatographic column, the sample injection amount is 5 μ l, methanol and 0.1% phosphoric acid solution in a volume ratio of 40:60 are used as mobile phases, the detection wavelength is 275nm, the column temperature is 18 ℃, and the volume flow is 1 mL/min; and in the step C, measuring the content of the indoleacetic acid standard substance solutions with different concentrations by using a high performance liquid chromatograph, drawing a standard curve, measuring by using the high performance liquid chromatograph to obtain the area of the indoleacetic acid peak in the sample solution to be measured, and calculating the indoleacetic acid content in the object to be measured by using a standard curve method.
The invention has the positive effects that:
(1) the invention synthesizes a novel indoleacetic acid adsorbent, which has the following principle: the nitrogen atom in the 2-vinyl quinoline is utilized to easily adsorb protons to form cations to be combined with the indole acetic acid (or indole acetate) with negative charge. Freezing, slicing, extracting and adsorbing a plant sample to be detected, adsorbing and enriching the indoleacetic acid by using an adsorbent, then releasing the indoleacetic acid by treatment, and quantitatively detecting the indoleacetic acid by adopting a high performance liquid chromatography. The detection method can enrich ppt-level indoleacetic acid, has high sensitivity, and provides a powerful tool means for safety evaluation of agricultural products.
(2) The method for detecting the indoleacetic acid in the invention adopts a frozen slicing method instead of a crushing and homogenizing method, so that the trace indoleacetic acid is not easy to run off, the extraction efficiency is improved, and the detection result is more accurate.
(3) According to the method for detecting the indoleacetic acid, the methanol is used for extracting the indoleacetic acid in the pretreatment of the agricultural products, so that the interference of a large amount of biomolecules such as protein, amino acid, fatty acid, starch, fiber and the like can be avoided, and the extraction efficiency of the indoleacetic acid is improved.
Drawings
FIG. 1 is an HPLC chromatogram for determining indoleacetic acid in kiwi fruit according to the detection method of example 1.
Detailed Description
Example 1
The method for detecting indoleacetic acid of the embodiment comprises the following specific steps:
A. preparing an indoleacetic acid adsorbent:
mu.l of 2-vinylquinoline (1.77mmol as functional monomer), 3.2ml of trimethylolpropane trimethacrylate (8.86mmol as crosslinker) and 0.041g of azobiphenyl (0.25mmol as free-radical initiator) were dissolved in 10ml of methanol and degassed under a nitrogen atmosphere. The mixture was purged with nitrogen to remove oxygen that inhibited polymerization. The glass tube was sealed and heated for thermal polymerization at 60 ℃ for 20 hours to give a yellow powder (the polymer thus produced expands in the solvent), ground in a mortar and dried under vacuum until constant weight. Subsequently, the polymer was washed 6 times with a detergent formulated from methanol and acetic acid (volume ratio 9: 1) to remove unreacted monomers and by-products during the synthesis to give an indoleacetic acid adsorbent.
B. Preparing a sample solution to be tested:
the pretreatment is to slice the kiwi fruits and then freeze the sliced kiwi fruits for 24 hours at the temperature of minus 20 ℃. Adding 1ml of 80% methanol into 20mg of lyophilized fructus Actinidiae chinensis sample, stirring for 30min, and centrifuging (5000r/min, 4 deg.C, 30 min). After centrifugation, the supernatant was transferred to a test tube and part of the methanol was removed under nitrogen. An appropriate amount of water was added to bring the methanol concentration to about 10%. This solution was added to 30mg of the prepared polymer. The resulting suspension was stirred at room temperature for 5min, centrifuged (5000r/min, 20 ℃, 10min) and the supernatant removed. Additional 1ml of 10% strength methanol was added and washed several times to remove the final interfering compounds. The polymer was treated with 1ml of 100% strength methanol to liberate the indoleacetic acid substance and the resulting suspension was stirred at room temperature for 5min and then centrifuged (5000r/min, 20 ℃ C., 10 min). The supernatant was collected using a pinhole filter device (0.25 μm). Under the nitrogen flowing condition, the removal rate of methanol was 50%. And adding a proper amount of water to restore the initial volume to obtain a sample solution to be detected.
C. Determining the solution of the sample to be tested:
and (3) formulating a standard curve, accurately measuring the standard solution, diluting the standard solution to 5 different concentrations step by step, and performing quantitative determination on the standard solution by using a high-performance liquid chromatograph. And drawing a standard curve by taking the mass concentration as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a regression equation and a correlation coefficient. Indoleacetic acid Y54518 x-20675, R2＝0.9999。
A150 mm multiplied by 2.1mm, 3 μm Thermo Acclaim C30 chromatographic column is adopted, the sample amount is 5 μ l, methanol and 0.1 percent phosphoric acid solution in the volume ratio of 40:60 are taken as mobile phases, the detection wavelength is 275nm, the column temperature is 18 ℃, and the volume flow is 1 mL/min.
And (3) quantitatively determining the solution of the sample to be detected on a high performance liquid chromatograph to obtain the peak area of the indoleacetic acid, and then obtaining the content of the indoleacetic acid in the sample to be detected according to the standard curve of the indoleacetic acid. And taking the mixed reference substance, carrying out continuous sample injection for 6 times, recording peak areas, and respectively recording the result peak areas RSD of 1.6% and 1.7%, thereby indicating that the precision of the instrument is good.
FIG. 1 is an HPLC chart of indole acetic acid assay of kiwi fruit samples using the indole acetic acid assay of this example. The peak with retention time of 13.186min in the figure is the chromatographic peak of indole acetic acid. The detection result has symmetrical peak pattern, good stability, high repeatability, high separation degree, low detection limit, high sensitivity and no damage to instruments. The HPLC chart effectively shows that the polymer adsorbent prepared by the research can selectively adsorb the indoleacetic acid.
Example 2
The method for detecting indoleacetic acid of the embodiment comprises the following specific steps:
A. preparing an indoleacetic acid adsorbent:
380. mu.l of 2-vinylquinoline (3.54mmol as functional monomer), 3.2ml of trimethylolpropane trimethacrylate (8.86mmol as crosslinker) and 0.082g of azobiphenyl (0.5mmol as free-radical initiator) are dissolved in 20ml of methanol and degassed under a nitrogen atmosphere. The mixture was purged with nitrogen to remove oxygen that inhibited polymerization. The glass tube was sealed and heated for thermal polymerization at 70 ℃ for 24 hours to give a yellow powder, which was ground in a mortar and dried under vacuum until constant weight. Subsequently, the polymer was washed 6 times with a detergent formulated from methanol and acetic acid (volume ratio 10: 1) to remove unreacted monomers and by-products during the synthesis to give an indoleacetic acid adsorbent.
B. Preparing a sample solution to be tested:
the pretreatment is to slice the kiwi fruits and then freeze the sliced kiwi fruits for 24 hours at the temperature of minus 20 ℃. Adding 1ml of 80% methanol into 20mg of lyophilized fructus Actinidiae chinensis sample, stirring for 30min, and centrifuging (5000r/min, 4 deg.C, 30 min). After centrifugation, the supernatant was transferred to a test tube and part of the methanol was removed under nitrogen. An appropriate amount of water was added to bring the methanol concentration to about 10%. This solution was added to 30mg of the prepared polymer. The resulting suspension was stirred at room temperature for 5min, centrifuged (5000r/min, 20 ℃, 10min) and the supernatant removed. Additional 1ml of 10% strength methanol was added and washed several times to remove the final interfering compounds. The polymer was treated with 1ml of 100% strength methanol to liberate the indoleacetic acid substance and the resulting suspension was stirred at room temperature for 5min and then centrifuged (5000r/min, 20 ℃ C., 10 min). The supernatant was collected using a pinhole filter device (0.25 μm). Under the nitrogen flowing condition, the removal rate of methanol was 50%. And adding a proper amount of water to restore the initial volume to obtain a sample solution to be detected.
C. Determining the solution of the sample to be tested:
and (3) formulating a standard curve, accurately measuring the standard solution, diluting the standard solution to 5 different concentrations step by step, and performing quantitative determination on the standard solution by using a high-performance liquid chromatograph. And drawing a standard curve by taking the mass concentration as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a regression equation and a correlation coefficient. Indoleacetic acid Y54518 x-20675, R2＝0.9999。
A150 mm multiplied by 2.1mm, 3 μm Thermo Acclaim C30 chromatographic column is adopted, the sample amount is 5 μ l, methanol and 0.1 percent phosphoric acid solution in the volume ratio of 40:60 are taken as mobile phases, the detection wavelength is 275nm, the column temperature is 18 ℃, and the volume flow is 1 mL/min.
And (3) quantitatively determining the solution of the sample to be detected on a high performance liquid chromatograph to obtain the peak area of the indoleacetic acid, and then obtaining the content of the indoleacetic acid in the sample to be detected according to the standard curve of the indoleacetic acid.
Example 3
The method for detecting indoleacetic acid of the embodiment comprises the following specific steps:
A. preparing an indoleacetic acid adsorbent:
mu.l of 2-vinylquinoline (2.655mmol, as functional monomer), 3.2ml of trimethylolpropane trimethacrylate (8.86mmol, as crosslinker) and 0.164g of azobiphenyl (1mmol, as free-radical initiator) were dissolved in 15ml of methanol and degassed under a nitrogen atmosphere. The mixture was purged with nitrogen to remove oxygen that inhibited polymerization. The glass tube was sealed and heated for thermal polymerization at 80 ℃ for 18 hours to give a yellow powder, which was ground in a mortar and dried under vacuum until constant weight. Subsequently, the polymer was washed 6 times with a detergent formulated from methanol and acetic acid (volume ratio 15: 1) to remove unreacted monomers and by-products during the synthesis to give an indoleacetic acid adsorbent.
B. Preparing a sample solution to be tested:
the pretreatment is to slice the kiwi fruits and then freeze the sliced kiwi fruits for 24 hours at the temperature of minus 20 ℃. Adding 1ml of 80% methanol into 20mg of lyophilized fructus Actinidiae chinensis sample, stirring for 30min, and centrifuging (5000r/min, 4 deg.C, 30 min). After centrifugation, the supernatant was transferred to a test tube and part of the methanol was removed under nitrogen. An appropriate amount of water was added to bring the methanol concentration to about 10%. This solution was added to 30mg of the prepared polymer. The resulting suspension was stirred at room temperature for 5min, centrifuged (5000r/min, 20 ℃, 10min) and the supernatant removed. Additional 1ml of 10% strength methanol was added and washed several times to remove the final interfering compounds. The polymer was treated with 1ml of 100% strength methanol to liberate the indoleacetic acid substance and the resulting suspension was stirred at room temperature for 5min and then centrifuged (5000r/min, 20 ℃ C., 10 min). The supernatant was collected using a pinhole filter device (0.25 μm). Under the nitrogen flowing condition, the removal rate of methanol was 50%. And adding a proper amount of water to restore the initial volume to obtain a sample solution to be detected.
C. Determining the solution of the sample to be tested:
and (3) formulating a standard curve, accurately measuring the standard solution, diluting the standard solution to 5 different concentrations step by step, and performing quantitative determination on the standard solution by using a high-performance liquid chromatograph. And drawing a standard curve by taking the mass concentration as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a regression equation and a correlation coefficient. Indoleacetic acid Y54518 x-20675, R2＝0.9999。
A150 mm multiplied by 2.1mm, 3 μm Thermo Acclaim C30 chromatographic column is adopted, the sample amount is 5 μ l, methanol and 0.1 percent phosphoric acid solution in the volume ratio of 40:60 are taken as mobile phases, the detection wavelength is 275nm, the column temperature is 18 ℃, and the volume flow is 1 mL/min.
And (3) quantitatively determining the solution of the sample to be detected on a high performance liquid chromatograph to obtain the peak area of the indoleacetic acid, and then obtaining the content of the indoleacetic acid in the sample to be detected according to the standard curve of the indoleacetic acid.
The reagents used in the present invention are chemically pure at concentrations not otherwise specified. The CAS number for 2-vinylquinoline is 772-03-2. Trimethylolpropane trimethacrylate has a CAS registry number of 3290-92-4 and azodiphenylene has a CAS registry number of 103-33-3.
In order to verify the effect of the indole acetic acid adsorbent, the indole acetic acid adsorbents in examples 1 to 3 were tested repeatedly by using an indole acetic acid standard, wherein the binding rate was calculated by detecting the content of indole acetic acid in the remaining solution after the indole acetic acid adsorbent was adsorbed, the release rate was calculated by detecting the content of indole acetic acid in the solution formed by the indole acetic acid adsorbent releasing indole acetic acid, and the test results are shown in table 1.
TABLE 1 binding and Release Effect of Indolylacetic acid adsorbents
| Indole acetic acid binding Rate (%) | Indoleacetic acid release rate (%) | |
| Example 1 | 96±3 | 71±2 |
| Example 2 | 95±4 | 73±1 |
| Example 3 | 94±3 | 72±2 |
As can be seen from Table 1, the binding rate of the indole acetic acid adsorbent polymer of the present invention to indole acetic acid is very high, about 95%, and about 70% of the indole acetic acid conjugate is released by methanol. No significant difference was observed between the two examples, indicating that the polymer properties were reproducible.
It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.
Claims (10)
1. A preparation method of indoleacetic acid adsorbent is characterized in that 2-vinylquinoline, trimethylolpropane trimethacrylate and azodiphenyl are dissolved in methanol, and are heated in a closed manner for thermal polymerization reaction to obtain yellow powder; then, the powder is washed with methanol and acetic acid to remove unreacted monomers and byproducts in the synthesis process, and a finished product is obtained.
2. The preparation method of the indoleacetic acid adsorbent as claimed in claim 1, wherein the molar ratio of 4-2-vinylquinoline to trimethylolpropane trimethacrylate to azodiphenyl is [ 4-2 ]: [ 10-8 ]: [ 0.1-1 ].
3. The method for preparing the indoleacetic acid adsorbent according to claim 2, wherein the temperature of the thermal polymerization is 60 to 100 ℃, and the time of the thermal polymerization is 10 to 30 hours.
4. The preparation method of the indoleacetic acid adsorbent as claimed in claim 2, wherein the powder is washed with a detergent prepared from methanol and acetic acid at a volume ratio of 5: 1 to 100: 1.
5. An indoleacetic acid adsorbent prepared by the process according to claim 1.
6. Use of the indole acetic acid adsorbent of claim 5 for the detection of residual indole acetic acid in agricultural products.
7. The method for detecting the indoleacetic acid is characterized by comprising the following steps of:
A. preparing an indoleacetic acid adsorbent: 2-vinyl quinoline and trimethylolpropane trimethacrylate are subjected to a cross-linking reaction to generate an indoleacetic acid adsorbent;
B. preparing a sample solution to be tested: freezing, slicing, extracting and adsorbing the agricultural product, mixing the agricultural product with the indoleacetic acid adsorbent to ensure that the indoleacetic acid adsorbent absorbs and enriches indoleacetic acid, and treating the indoleacetic acid adsorbent with methanol to ensure that the indoleacetic acid adsorbent releases indoleacetic acid to obtain a sample solution to be detected;
C. determining the solution of the sample to be tested: taking the sample solution to be measured, and carrying out quantitative determination by a high performance liquid chromatograph.
8. The method for detecting indoleacetic acid as claimed in claim 7, wherein the preparation method of the indoleacetic acid adsorbent in step A is: dissolving 2-vinylquinoline, trimethylolpropane trimethacrylate and azodiphenyl in methanol, sealing and heating for thermal polymerization reaction to obtain yellow powder; then, the powder is washed with methanol and acetic acid to remove unreacted monomers and byproducts in the synthesis process, and a finished product is obtained.
9. The method for detecting indoleacetic acid as claimed in claim 7, wherein the step B of pretreating the agricultural product comprises the steps of: slicing and freezing an agricultural product, adding methanol into a freeze-dried agricultural product sample, stirring and centrifuging, taking supernate, transferring the supernate into a test tube, and adding the supernate into an indoleacetic acid adsorbent; stirring the obtained suspension at room temperature, centrifuging, and removing supernatant; adding methanol for washing for many times to remove the final interfering compound; then, treating the indoleacetic acid adsorbent with methanol with the concentration of 100% to release indoleacetic acid, stirring the obtained suspension at room temperature, centrifuging, and filtering supernatant to obtain a sample solution to be detected.
10. The method for detecting indoleacetic acid as claimed in claim 7, wherein in step C, the HPLC detection method is 150mm x 2.1mm, 3 μm Thermo Acclaim C30 chromatographic column, the sample amount is 5 μ l, methanol and 0.1% phosphoric acid solution in a volume ratio of 40:60 are used as mobile phase, the detection wavelength is 275nm, the column temperature is 18 ℃, and the volume flow is 1 mL/min; and in the step C, measuring the content of the indoleacetic acid standard substance solutions with different concentrations by using a high performance liquid chromatograph, drawing a standard curve, measuring by using the high performance liquid chromatograph to obtain the area of the indoleacetic acid peak in the sample solution to be measured, and calculating the indoleacetic acid content in the object to be measured by using a standard curve method.
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