CN110618212B - Method for simultaneously detecting residual quantity of multiple plant growth regulators in vegetables - Google Patents
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Abstract
The invention discloses a method for simultaneously detecting the residual quantity of multiple plant growth regulators in vegetables, which can simultaneously detect three commonly used plant growth regulators, namely 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin, and has the advantages of high specificity, sensitivity and stability, simple operation and lower detection limit. By adjusting various parameters in the existing detection technology, particularly optimizing the solvent, conditions and the like adopted by the mobile phase and the sample during pretreatment, the detection process is simplified, the detection time is saved, and the method is more suitable for large-batch detection.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for simultaneously detecting residual quantities of various plant regulators in vegetables.
Background
Plant growth regulators (plant growth regulators) are substances which have similar physiological and biological effects to plant hormones and are synthesized or extracted to have similar physiological and biological effects to plant hormones, and are pesticides for regulating plant growth and development, including artificially synthesized compounds and natural plant hormones extracted from organisms, such as gibberellin, 2, 4-D, mepiquat chloride and the like, and are widely applied to the aspects of regulating the growth of fruits and vegetables, improving the quality and yield of crops and the like.
However, while bringing great economic benefits to agricultural production, cases of abuse and improper use of plant growth regulators frequently occur. For example, abnormal strawberries which are excessively ripened, mangoes which are ripened by ripener, bean sprouts which are soaked in rootless water and the like, excessive plant growth regulators ingested by human bodies can stimulate skin mucosa, and the phenomena of nausea, vomiting and the like occur, and the long-term use of the plant growth regulators has certain accumulated toxicity.
Especially, bean sprouts are vegetables with large daily edible amount, and in 2015, the general administration of food and drug administration, the ministry of agriculture, the national health and family planning committee also issues a notice about prohibition of using substances such as 6-benzyladenine and the like in the bean sprout production process (No. 11 in 2015), and the following are clearly specified: 6-benzyl adenine, 4-sodium chlorophenoxyacetate, gibberellin and other substances are used as low-toxicity pesticides, registered and managed, the application range is limited, in order to ensure the edible safety of the bean sprouts, producers cannot use the 6-benzyl adenine, the 4-sodium chlorophenoxyacetate, the gibberellin and other substances in the bean sprout production process, and bean sprout operators cannot operate the bean sprouts containing the 6-benzyl adenine, the 4-sodium chlorophenoxyacetate, the gibberellin and other substances. In order to ensure the health of people, it is very important to establish an accurate and reliable test method.
The existing instrumental analysis and detection method for the plant growth regulator in the bean sprouts comprises a liquid chromatography and a liquid chromatography tandem mass spectrometry, the detection result is not very accurate, the detection time is long, the pretreatment process is relatively complex, the time consumption is long, and the detection limit is high. Although the measurement of the plant growth regulator in the bean sprouts of BJS 201703 by using a liquid chromatography-mass spectrometer is disclosed in "measurement of the plant growth regulator in the bean sprouts of 11", the inventors have carried out the measurement by this method, the detection limit is high, the pretreatment step is complicated, and the detection takes a long time.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a method for simultaneously detecting the residual quantity of various plant growth regulators in vegetables, which can simultaneously detect three commonly used plant growth regulators, and has the advantages of high specificity, sensitivity and stability, simple operation and lower detection limit.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for simultaneously detecting the residual quantity of multiple plant growth regulators in vegetables, wherein the plant growth regulators are 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin, and the method comprises the following steps:
(1) pretreatment of a sample to be detected:
A. extraction of
Weighing 5.00g of sample in a 50mL centrifuge tube, adding 20mL of 0.01mol/L sodium hydroxide solution, uniformly mixing for 5min in a vortex manner, centrifuging for 5min at 5000r/min, and taking 10mL of supernatant in another 50mL centrifuge tube;
B. purification
Adding 2mL of hydrochloric acid aqueous solution with volume fraction of 50% into 10mL of supernate, mixing uniformly, adding 20mL of ethyl acetate for extraction, mixing uniformly for 5min in a vortex manner, centrifuging for 5min at 5000r/min, sucking all supernate into a 50mL centrifuge tube, and drying in a water bath at 40 ℃ by nitrogen;
C. redissolving
Adding 1mL of methanol into the dried centrifugal tube, performing vortex dissolution, and filtering through a 0.22-micron filter membrane to obtain a filtrate containing the plant growth regulator, namely a sample to be detected, to be analyzed;
(2) preparing a standard solution:
respectively weighing 10mg of 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin standard substance in a 10mL volumetric flask, respectively preparing 1000 mug/mL standard stock solutions by using methanol, respectively transferring the standard stock solutions to prepare a mixed standard substance reference solution with the concentration of 10 mug/mL; then preparing standard stock solution into standard substance solutions with the concentrations of 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 800ng/mL respectively for later use by using a blank matrix solution when a standard curve is prepared;
(3) carrying out qualitative and quantitative detection on the sample to be detected by using the pretreated sample through a liquid chromatography-mass spectrometer to obtain the content of the plant growth regulator of the sample to be detected, and specifically comprising the following steps:
A. sampling 5.0 mu L of sample solution to be detected, and analyzing and detecting by using a liquid chromatography-mass spectrometer to obtain a total ion current chromatogram, a quantitative ion pair chromatogram and a qualitative ion pair chromatogram of the sample solution to be detected;
B. analyzing and detecting the standard curve solutions with different concentrations prepared in the step (2) by using a liquid chromatography-mass spectrometer, wherein the detection conditions are the same as those of the sample solution to be detected, and obtaining a total ion current chromatogram, a quantitative ion chromatogram and a qualitative ion pair chromatogram of the standard solution;
C. preparing a standard curve of the plant growth regulator according to the chromatographic peak areas of the quantitative ion pair and the qualitative ion pair of the substance to be measured;
D. according to the chromatographic peaks of the quantitative ion pairs and the qualitative ion pairs of the plant growth regulator in the sample solution to be detected, the concentration C of the plant growth regulator in the sample solution to be detected is calculated by combining a standard curve, the content X of the plant growth regulator in the vegetable sample is calculated according to the following formula, and the content calculation formula is as follows:
X＝C*V*f*1000/(m*1000)
wherein X is the content of the plant growth regulator in the sample to be detected, and the unit is mu g/kg; c is the concentration of the plant growth regulator in the sample to be detected, and the unit is ng/mL; v is the dissolving volume of the sample to be detected, and the unit is mL; f is the pretreatment dilution multiple, and f is 2 in the experiment; and m is the mass of the sample to be measured and is expressed in g.
Specifically, the conditions for detecting the sample to be detected by the liquid chromatography-mass spectrometer are as follows: the mobile phase is a mixed aqueous solution of methanol and 5mmol/L ammonium acetate, the volume ratio of the two is (20-90): (10-80), gradient elution is carried out, the elution time is 6min, and the flow rate of the mobile phase is 0.4 mL/min; the chromatographic column is an octadecylsilane chromatographic column, the size of the chromatographic column is 3.0 x 150mm x 2.7 microns; the sample size for detection was 5 μ L, column temperature: at 30 ℃. The gradient elution details are shown in table 1:
TABLE 1 gradient elution
The mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument during detection are as follows: ionization mode: electrospray ionization negative ion mode; the scanning mode is as follows: multiple Reaction Monitoring (MRM); ejection voltage: 2000V; delta EM: 200V; atomizer pressure: 40 psi; flow rate of drying gas: 10L/min; temperature of the drying gas: 345 deg.C.
Wherein the vegetable sample is a vegetable which may contain a plant growth regulator, preferably bean sprouts.
The invention has the beneficial effects that:
the detection method has the advantages of short detection time, high sensitivity, strong selectivity, good repeatability and simple operation.
The invention simplifies the detection process by adjusting various parameters in the prior detection technology, particularly optimizing the solvent, conditions and the like adopted by the treatment of the mobile phase and the sample during the pretreatment.
Selecting the mobile phase for detection as methanol and 5mmol/L ammonium acetate water solution; furthermore, the method can simultaneously determine the 6-benzyladenine, the 4-sodium chlorophenoxyacetate and the gibberellin in the plant growth regulator, and has the advantages of high specificity, high sensitivity and lower detection limit. The inventors also specifically compared the method of the present invention with the following two methods:
compared with the determination of the residual quantity of the p-chlorophenoxyacetic acid in SN/T3725-2013 export food, the method has the advantages that:
in the invention, target chemicals are extracted by alkali solution, a sample to be detected is prepared by purifying the sample by a liquid-liquid extraction method, and qualitative and quantitative determination is carried out by a liquid-phase mass spectrometer. Compared with the prior art, the method has the advantages of fewer operation steps and smaller system error; by means of mass spectrum multiple reaction monitoring, three auxin regulators of 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin can be measured simultaneously, the detection limit is lower, the detection efficiency is higher, and the method is more suitable for actual detection work of a large number of samples and multiple detection targets.
Compared with the determination of the plant growth regulator in the BJS 201703 bean sprouts, the method has the advantages that:
in the invention, the required dosage for detecting each sample is 5g, only sodium hydroxide solution is needed for extracting the reagent, and the purification step is a liquid-liquid extraction method; when the method is used for measurement by using a liquid phase mass spectrometer, the time for the method is only 30 minutes, which is one half of 'measurement of plant growth regulators in BJS 201703 bean sprouts', and the efficiency is extremely high; the method can detect 3 common plant growth regulators, namely 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin, relatively specifically, is more suitable for conventional sample detection compared with the method for simultaneously detecting 11 plant growth regulators, and has the advantages of strong representativeness, flexibility and high efficiency.
The 3 plant growth regulators of 6-benzyladenine, sodium 4-chlorophenoxyacetate and gibberellin were detected according to the pretreatment method in the method "determination of plant growth regulators in BJS 201703 Bean sprouts", and compared with the present invention: the 20 batches of samples are respectively detected, the former pretreatment time is about 142min, the invention only needs 84min, the time is saved by one half, the detection limit is higher than that of the invention, the sensitivity is poor, the detection time on the computer is 24min, the time on the computer is only 6min, and two thirds of the time is saved.
Drawings
FIG. 1 is a total ion flow diagram of 4-chlorophenoxy sodium acetate, 6-benzyladenine and gibberellin standard substances in a sample solution;
FIG. 2 is a total ion flow diagram of 4-chlorophenoxy sodium acetate, 6-benzyladenine and gibberellin standard substances in a standard control solution;
FIG. 3 is a signal-to-noise ratio spectrum of 4-chlorophenoxysodium acetate in a sample solution
FIG. 4 is a spectrum of the noise ratio of 6-benzyladenine in a sample solution;
FIG. 5 is a spectrum of the noise ratio of gibberellin in a sample solution;
FIG. 6 is a standard curve of sodium 4-chlorophenoxyacetate;
FIG. 7 is a standard curve for 6-benzyladenine;
FIG. 8 is a standard curve for gibberellins;
FIG. 9 is a quantitative ion-pair chromatogram of sodium 4-chlorophenoxyacetate in a standard control solution;
FIG. 10 is a quantitative ion pair chromatogram of 6-benzyladenine in a standard control solution;
FIG. 11 is a quantitative ion pair chromatogram of gibberellin in a standard control solution;
FIG. 12 is a quantitative, qualitative ion pair abundance colorimetric spectrum of sodium 4-chlorophenoxyacetate in standard control solution;
FIG. 13 is a colorimetric spectrum of quantitative qualitative ion pair abundance of 6-benzyladenine in standard control solution;
FIG. 14 is a colorimetric spectrum of quantitative, qualitative ion pair abundance of gibberellins in a standard control solution;
Detailed Description
The invention will be further explained by means of specific embodiments, however, it should be understood that the invention may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
First, test
1. Material
1.1 Instrument: agilent, liquid chromatography-mass spectrometer 1290 II-6460.
1.2 reagent: water (first-order water), methanol (chromatographically pure), ethyl acetate (chromatographically pure), hydrochloric acid (chemically pure), ammonium acetate (chemically pure), sodium hydroxide (chemically pure), and water for preparing the solution are all first-order water.
1.3 comparison products: 6-benzyladenine (CAS: 1214-39-7), sodium 4-chlorophenoxyacetate (CAS: 13730-98-8), gibberellin (CAS: 77-06-5).
1.4 sample: bean sprouts (purchased from local vegetable markets).
2. Method and results
2.1 detection conditions
2.1.1 sample introduction conditions
A chromatographic column: octadecylsilane column (3.0 x 150mm x 2.7 μm);
mobile phase: 5mmol/L ammonium acetate aqueous solution and methanol, corresponding to the volume ratio at different times shown in Table 1;
sample introduction amount: 5 mu L of the solution;
flow rate: 0.4 mL/min;
column temperature: at 30 ℃.
2.1.2 Mass Spectrometry conditions
Ionization mode: electrospray ionization negative ion mode;
the scanning mode is as follows: multiple Reaction Monitoring (MRM);
ejection voltage: 2000 v;
delta EM (Δ -electromagnetic collision): 200V;
atomizer pressure: 40 psi;
flow rate of drying gas: 10L/min;
temperature of the drying gas: 345 deg.C.
Therein, the monitored ion pairs and their corresponding collision energies are shown in table 2.
Table 2 monitoring ion pairs and their corresponding collision energies
3. Test procedure
(1) Pretreatment of a sample to be detected:
A. extraction of
Weighing 5.00g of sample in a 50mL centrifuge tube, adding 20mL of 0.01mol/L sodium hydroxide solution, uniformly mixing for 5min in a vortex manner, centrifuging for 5min at 5000r/min, and taking 10mL of supernatant in another 50mL centrifuge tube;
B. purification
Adding 2mL of hydrochloric acid aqueous solution with volume fraction of 50% into 10mL of supernate, mixing uniformly, adding 20mL of ethyl acetate for extraction, mixing uniformly for 5min in a vortex manner, centrifuging for 5min at 5000r/min, sucking all supernate into a 50mL centrifuge tube, and drying in a water bath at 40 ℃ by nitrogen;
C. redissolving
Adding 1mL of methanol into the dried centrifugal tube, performing vortex dissolution, and filtering through a 0.22-micron filter membrane to obtain a filtrate containing the plant growth regulator, namely a sample to be detected, to be analyzed;
(2) preparing a standard solution:
respectively weighing 10mg of 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin standard substance in 10mL volumetric flasks, respectively preparing 1000 mug/mL standard stock solutions, respectively transferring the standard stock solutions to prepare a mixed standard substance reference solution with the concentration of 10 mug/mL.
And (3) sucking a proper amount of mixed standard substance control solution according to experiment needs, diluting the mixed standard substance control solution into standard working solution with proper concentration by using the matrix blank solution, and preparing the working solution on site.
(3) And (3) quantitatively analyzing the pretreated sample by using a liquid chromatography-mass spectrometer, and qualitatively and quantitatively detecting the sample to be detected to obtain the content of the plant growth regulator in the vegetable sample.
A. Sampling 5.0 μ L of sample solution to be detected, analyzing and detecting with triple quadrupole liquid chromatography-mass spectrometer to obtain total ion current chromatogram (see figure 1), quantitative ion pair chromatogram and qualitative ion pair relative abundance chromatogram of plant growth regulator in the sample solution to be detected, and calculating with instrument to obtain signal-to-noise ratio chromatogram (see figures 3-5);
B. analyzing and detecting the standard curve solutions with different concentrations prepared in the step (2) by using a triple quadrupole liquid chromatography-mass spectrometer, wherein the detection conditions are the same as those of the sample solution to be detected, and obtaining a total ion current chromatogram (shown in figure 2), a quantitative ion chromatogram and a qualitative ion pair relative abundance colorimetric chromatogram of the standard solution;
C. preparing a standard curve of the plant growth regulator according to the chromatographic peak areas of the quantitative ion pair and the qualitative ion pair of the plant growth regulator to be detected (see the figure 6-figure 8); the quantitative ion chromatogram of the sodium 4-chlorophenoxyacetate is shown in figure 9, the relative abundance chromatogram of the quantitative and qualitative ion pair is shown in figure 12, and the standard curve is shown in figure 6; the 6-benzyladenine quantitative ion chromatogram is shown in figure 10, the quantitative qualitative ion pair relative abundance chromatogram is shown in figure 13, the standard curve is shown in figure 7, the gibberellin quantitative ion chromatogram is shown in figure 11, the quantitative qualitative ion pair relative abundance chromatogram is shown in figure 14, and the standard curve is shown in figure 8.
D. According to the chromatographic peaks of the quantitative ion pairs and the qualitative ion pairs of the plant growth regulator in the sample solution to be detected, the concentration C of the plant growth regulator in the sample solution to be detected is calculated by combining a standard curve, the content X of the plant growth regulator in the vegetable sample is calculated according to the following formula, and the content calculation formula is as follows:
X＝C*V*f*1000/(m*1000)
wherein X is the content of the plant growth regulator in the sample to be detected, and the unit is mu g/kg; c is the concentration of the plant growth regulator in the sample to be detected, and the unit is ng/mL; v is the dissolving volume of the sample to be detected, and the unit is mL; f is the pretreatment dilution multiple, and f is 2 in the experiment; and m is the mass of the sample to be measured and is expressed in g.
The mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument are as follows:
ionization mode: electrospray ionization negative ion mode; the scanning mode is as follows: multiple Reaction Monitoring (MRM); ejection voltage: 2000V; delta EM: 200V; atomizer pressure: 40 psi; flow rate of drying gas: 10L/min; temperature of the drying gas: 345 deg.C.
4. Test results and analysis
4.1 detection Limit
And selecting a low-concentration point for sample injection, calculating a corresponding signal-to-noise ratio, and comparing the signal-to-noise ratio with a reported detection limit. The signal to noise ratio calculations are shown in table 3.
TABLE 3 SNR calculation results
As can be seen from FIGS. 3-5 and Table 2: when the injection concentration of the 4-sodium chlorophenoxyacetate, the 6-benzyladenine and the gibberellin is 20ng/ml, the signal-to-noise ratios are 177.8, 1485.1 and 98.8 respectively, and the requirements are met.
4.2 linearity of the Standard Curve
The measurement was carried out according to the above-mentioned detection method using a standard working curve prepared with a blank medium at a concentration of 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL or 800 ng/mL.
From FIGS. 6-8, it follows that: the linear correlation coefficient of the standard curves of the 4-sodium chlorophenoxyacetate, the 6-benzyladenine and the gibberellin is more than 0.99, and the requirements are met.
4.3 recovery and accuracy
For forbidden substances, the determination lower limit, the two-time method determination lower limit and the ten-time method determination lower limit are selected to be added in three levels, the repeated determination times are 6, namely, the bean sprout samples are subjected to three-level six-parallel detection by respectively performing labeling determination with 8 mu g/kg, 16 mu g/kg and 80 mu g/kg, and the determination results are shown in table 4.
TABLE 4 results of three-level six-parallel assay
As can be seen from the table, the recovery rates of the three different levels of addition recovery tests are all over 90 percent, the recovery rates meet the requirements, the same level is subjected to 6 times of parallel measurement, the calculated relative standard deviation is less than 10 percent, and the repeatability of the method meets the requirements.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.
Claims (5)
1. A method for simultaneously detecting the residual quantity of multiple plant growth regulators in vegetables is characterized in that the plant growth regulators are 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin, and the method comprises the following steps:
(1) pretreatment of a sample to be detected:
A. extraction of
Weighing 5.00g of sample in a 50mL centrifuge tube, adding 20mL of 0.01mol/L sodium hydroxide solution, uniformly mixing for 5min in a vortex manner, centrifuging for 5min at 5000r/min, and taking 10mL of supernatant in another 50mL centrifuge tube;
B. purification
Adding 2mL of hydrochloric acid aqueous solution with volume fraction of 50% into 10mL of supernate, mixing uniformly, adding 20mL of ethyl acetate for extraction, mixing uniformly for 5min in a vortex manner, centrifuging for 5min at 5000r/min, sucking all supernate into a 50mL centrifuge tube, and drying in a water bath at 40 ℃ by nitrogen;
C. redissolving
Adding 1mL of methanol into the dried centrifugal tube, performing vortex dissolution, and filtering through a 0.22-micron filter membrane to obtain a filtrate containing the plant growth regulator, namely a sample to be detected, to be analyzed;
(2) preparing a standard solution:
respectively weighing 10mg of 6-benzyladenine, 4-sodium chlorophenoxyacetate and gibberellin standard substances into 10mL volumetric flasks, respectively preparing 1000 mug/mL standard stock solutions by using methanol, respectively transferring the standard stock solutions to prepare mixed standard substance reference solutions with the concentrations of 10 mug/mL; then preparing the mixed standard substance control solution into standard substance solutions with the concentrations of 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 800ng/mL respectively for later use by using a blank matrix solution when a standard curve is prepared;
(3) carrying out qualitative and quantitative detection on the sample to be detected by using a liquid chromatography-mass spectrometer to obtain the content of three plant growth regulators of the sample to be detected;
the conditions for detecting the sample to be detected by using the liquid chromatography-mass spectrometer are as follows: the mobile phase is a mixed aqueous solution of methanol and 5mmol/L ammonium acetate, gradient elution is carried out, the elution time is 6min, and the flow rate of the mobile phase is 0.4 mL/min;
the gradient elution details are shown in the following table:
2. the method for simultaneously detecting the residual quantity of the multiple plant growth regulators in the vegetables as claimed in claim 1, wherein the step (3) is as follows:
A. sampling 5.0 mu L of sample solution to be detected, and analyzing and detecting by using a liquid chromatography-mass spectrometer to obtain a total ion current chromatogram, a quantitative ion pair chromatogram and a qualitative ion pair chromatogram of the sample solution to be detected;
B. analyzing and detecting the standard solution with different concentrations prepared in the step (2) by using a liquid chromatography-mass spectrometer, wherein the detection conditions are the same as those of the sample solution to be detected, and obtaining a total ion current chromatogram, a quantitative ion chromatogram and a qualitative ion pair chromatogram of the standard solution;
C. preparing a standard curve of the plant growth regulator according to the chromatographic peak areas of the quantitative ion pair and the qualitative ion pair of the substance to be measured;
D. according to the chromatographic peaks of the quantitative ion pairs and the qualitative ion pairs of the plant growth regulator in the sample solution to be detected, the concentration C of the plant growth regulator in the sample solution to be detected is calculated by combining a standard curve, the content X of the plant growth regulator in the vegetable sample is calculated according to the following formula, and the content calculation formula is as follows:
X＝C*V*f*1000/(m*1000)
wherein X is the content of the plant growth regulator in the sample to be detected, and the unit is mu g/kg; c is the concentration of the plant growth regulator in the sample to be detected, and the unit is ng/mL; v is the dissolving volume of the sample to be detected, and the unit is mL; f is the pretreatment dilution multiple, and f is 2 in the experiment; and m is the mass of the sample to be measured and is expressed in g.
3. The method for simultaneously detecting the residual quantity of multiple plant growth regulators in vegetables according to claim 1, wherein the chromatographic column for detection by the liquid chromatography-mass spectrometer is an octadecylsilane chromatographic column, and the size of the chromatographic column is 3.0 x 150mm x 2.7 microns; the sample size for detection was 5 μ L, column temperature: at 30 ℃.
4. The method for simultaneously detecting the residual quantity of the multiple plant growth regulators in the vegetables as claimed in claim 1, wherein the mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument are as follows: ionization mode: electrospray ionization negative ion mode; the scanning mode is as follows: multiple Reaction Monitoring (MRM); ejection voltage: 2000V; delta EM: 200V; atomizer pressure: 40 psi; flow rate of drying gas: 10L/min; temperature of the drying gas: 345 deg.C.
5. The method for simultaneously detecting the residual quantity of multiple plant growth regulators in vegetables according to any one of claims 1 to 4, wherein the vegetables are bean sprouts.
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