Method for detecting residual quantity of 20 plant growth hormones in strawberries

Abstract

The invention belongs to the technical field of detection methods, and particularly relates to a method for detecting the residual quantity of 20 plant growth hormones in strawberries. The method specifically comprises the following steps: preparing a standard substance and preparing a standard solution: providing a plant growth hormone standard substance, and preparing the standard substance into the standard solution for later use; preparing a to-be-detected solution, namely weighing strawberries, taking edible parts, homogenizing, purifying to obtain supernate after treatment, and preparing into the to-be-detected solution for later use; test condition setting: setting a positive mode liquid chromatography condition and a negative mode liquid chromatography condition; and sample detection: performing chromatographic detection on the standard solution and the solution to be detected. According to the detection method provided by the invention, 20 plant growth hormones in strawberries can be detected at the same time, the accuracy of the detection method is reliable, the detection limit is 0.1 mg / kg, the detection requirements are met, and the method has popularization and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of detection methods, and more specifically relates to a method for detecting 20 plant growth hormone residues in strawberries. Background Art

[0002] As a widely grown fruit, strawberry may be regulated by various plant growth hormones during its growth and development. These hormones include auxin, cytokinin, etc., which play a key role in the growth, flowering, and fruiting of strawberries. However, in order to increase yield or improve quality, some growers may use exogenous hormones, such as growth enhancers, to treat strawberries. The residues of these hormones may have adverse effects on human health, so the detection of growth hormones in strawberries becomes particularly important.

[0003] Chinese invention patent CN103969363A discloses a method for detecting plant growth regulator residues in fruits. The disclosed patent records that the QuEChERS method is used to extract and purify the plant growth regulator residues in fruits by adding acetonitrile containing acetic acid, so that the spiked recovery rate of the target compound reaches 71%-98%, and the relative standard deviation is not higher than 6%. It has good sensitivity and selectivity, and the recovery rate is stable. UPLC-MS / MS is used to achieve simultaneous detection of chlorfenapyr, ethephon, sodium 4-chlorophenoxyacetate and sodium 2,4-dichlorophenoxyacetate. The method is simple, and the detection limit of the four target compounds reaches 0.5-100.0μg / kg.

[0004] However, the disclosed patent can only detect four types of growth hormones, which is not applicable to fruits such as strawberries that contain multiple types of growth hormones on the market. In addition, the detection method is complicated and has low value for promotion and application. Developing a method that can simultaneously detect the residues of multiple types of growth hormones is an important technological breakthrough. Summary of the invention

[0005] In order to solve the problems in the prior art, the first aspect of the present invention provides a method for detecting 20 plant growth hormone residues in strawberries, comprising the following steps:

[0006] Preparation of standard products and preparation of standard solutions: provide plant growth hormone standards and prepare the standards into standard solutions for standby use;

[0007] Preparation of the test solution: Weigh the strawberries, take the edible part, homogenize it, purify it after treatment to obtain the supernatant, and prepare it as the test solution for later use;

[0008] Test condition setting: setting positive mode liquid chromatography conditions and negative mode liquid chromatography conditions;

[0009] Sample testing: The standard solution and the test solution are subjected to chromatographic testing.

[0010] In some preferred embodiments, the step of obtaining the supernatant specifically includes: weighing the homogenized sample to be tested, adding a dissolving solution and a solid adsorbent, homogenizing, centrifuging, and then taking the upper solution, adding a solid phase extraction adsorbent, and centrifuging to obtain a supernatant.

[0011] In some preferred embodiments, the solid adsorbent includes at least one of anhydrous magnesium sulfate, anhydrous sodium acetate, anhydrous calcium chloride, and soda lime.

[0012] In some preferred embodiments, the solid adsorbent includes anhydrous magnesium sulfate and anhydrous sodium acetate.

[0013] In some preferred embodiments, the solid adsorbent includes anhydrous magnesium sulfate and anhydrous sodium acetate, and the weight ratio of the anhydrous magnesium sulfate to the anhydrous sodium acetate is (1-5):1.

[0014] In some preferred embodiments, the weight ratio of anhydrous magnesium sulfate to anhydrous sodium acetate is 4:1.

[0015] In some preferred embodiments, the solid phase extraction adsorbent is C 18 Solid Phase Extraction Adsorbents.

[0016] In some preferred embodiments, the C 18 The particle size of the solid phase extraction adsorbent is 40 to 60 μm.

[0017] In some preferred embodiments, the specific steps of preparing the test solution include: weighing strawberries, taking the edible part, homogenizing, weighing 5.00 g of the homogenized sample to be tested, placing it in a 50 mL centrifuge tube, adding 20 mL of acetonitrile solution (v / v) containing 1% acetic acid, 4.0 g of anhydrous magnesium sulfate, and 1.0 g of anhydrous sodium acetate, high-speed homogenization for 5 min, and centrifuging at 400 r / min for 5 min; then taking 5.0 mL of the upper solution and adding it to a 250 mg C 18 and 750 mg of anhydrous magnesium sulfate in a centrifuge tube, vortex for 30 seconds, centrifuge at 4000 r / min for 5 minutes, take 0.5 mL of the supernatant into a 2 mL microcentrifuge tube, add 0.5 mL of 0.1% formic acid aqueous solution and mix well, filter through a 0.2 μm microporous membrane to obtain the test solution for use.

[0018] The solutions used in this application are as follows:

[0019] The acetonitrile solution containing 1% acetic acid is specifically prepared as follows: 10 mL of acetic acid (superior grade) is added to a 1000 mL volumetric flask, and then 990 mL of acetonitrile is added to the scale, shaken well, and set aside.

[0020] The specific method of 0.1% formic acid aqueous solution is as follows: 1.0 mL of formic acid (HPLC grade) is added into a 1000 mL volumetric flask, and then water is added to make up to the scale line, shaken and set aside.

[0021] In some preferred embodiments, the growth hormone standard products specifically include 2.4.-D, S-abscisic acid, 6-benzyladenine, paclobutrazol, chlormequat, brassinolide, gibberellic acid, chlormequat, mepiquat, clopyralid, p-chlorophenoxyacetic acid (abscisic acid), naphthylacetic acid, indoleacetic acid, thidiazuron, uniconazole, butyric acid hydrazide, trinexapac-ethyl, ampicillin, 2,3,5-triiodobenzoic acid, and ethephon, a total of 20 types.

[0022] In some preferred embodiments, the method for preparing the standard solution comprises the following steps: weighing a plant growth hormone standard, using acetonitrile as a solvent, preparing a standard solution with a concentration of 1000 μg / mL, and storing it at -18°C away from light.

[0023] In some preferred embodiments, it also includes the preparation of a mixed standard solution, and the method for preparing the mixed standard solution includes the following steps: taking the above-prepared standard solution, preparing a mixed standard solution of 20 plant growth hormones with a concentration of 1.0 μg / mL with acetonitrile, and storing it at 0°C to 4°C away from light.

[0024] In some preferred embodiments, a mixed standard working solution is further prepared, and the method for preparing the mixed standard working solution comprises the following steps: aspirating the mixed standard solution, preparing mixed standard working solutions of various concentrations with blank sample matrix, and preparing them for immediate use.

[0025] Note: The blank sample matrix here refers to the extraction reagent that does not contain the target substance.

[0026] In some preferred embodiments, the positive mode liquid chromatography conditions include:

[0027] Mobile Phase:

[0028] Phase A: acetonitrile; Phase B: 0.1% formic acid aqueous solution

[0029] Flow rate: 0.1~0.5mL / min

[0030] Column temperature: 40°C

[0031] Injection volume: 10 μL

[0032] Chromatographic column: Agilent ZORBA×Eclipse Plus C18, 100×2.1mm, 3.5μm (or equivalent column)

[0033] Analysis time: 10-15 minutes

[0034] The mobile phase elution gradient is shown in Table 1 below

[0035] Table 1: Positive mode mobile phase elution gradient

[0036]

[0037] The flow rate of the positive mode liquid chromatography condition can be listed as 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, and 0.5 mL / min.

[0038] The analysis time of the positive mode liquid chromatography condition can be listed as 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0039] In some preferred embodiments, the negative mode liquid chromatography conditions include:

[0040] Mobile Phase:

[0041] Phase A: acetonitrile; Phase B: water

[0042] Flow rate: 0.1~0.5mL / min

[0043] Column temperature: 40°C

[0044] Injection volume: 10 μL

[0045] Chromatographic column: Agilent ZORBA×Eclipse Plus C18, 100×2.1mm, 3.5μm (or equivalent column)

[0046] Analysis time: 10-15 minutes

[0047] The mobile phase elution gradient is shown in Table 2 below

[0048] Table 2 Negative mode mobile phase elution gradient

[0049]

[0050] The flow rate of the negative mode liquid chromatography condition can be listed as 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, and 0.5 mL / min.

[0051] The analysis time of the negative mode liquid chromatography condition can be listed as 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0052] In this application, the negative mode mass spectrometry parameters of 11 growth hormones are shown in Table 3.

[0053] Table 3: Negative mode mass spectrometry parameters of 11 plant growth hormones

[0054]

[0055] Note: The ion pairs marked with * are quantitative ion pairs. In this application, the negative mode mass spectrometry parameters of 9 growth hormones are shown in Table 4.

[0056] Table 4: Positive mode mass spectrometry parameters of 9 plant growth hormones

[0057]

[0058] Note: The ion pairs marked with * are quantitative ion pairs.

[0059] The method for detecting 20 growth hormones in strawberries provided by the present invention can accurately detect the contents of 20 growth hormones in strawberries by combining the positive mode and the negative mode. The method is convenient and efficient, not only has high accuracy, but also can detect 20 growth hormones at the same time, subtracting the time of multiple detections, and can be promoted for use.

[0060] The detection method provided in the present invention uses a particle size of 40μm-60μmC 18 Solid phase extraction adsorbent can achieve complete adsorption of growth hormone in strawberries, ensuring that the accuracy of test data is further improved during the detection process.

[0061] The second aspect of the present invention provides an application of a method for detecting the residues of 20 plant growth hormones in strawberries, which is used for detecting growth hormones in fruits and vegetables.

[0062] Beneficial Effects

[0063] (1) The detection method provided by the present invention can simultaneously detect 20 plant growth hormones present in strawberries. The detection method has reliable accuracy and a detection limit of 0.1 mg / kg, which meets the detection requirements and has the value of being promoted and applied;

[0064] (2) The detection method provided by the present invention can realize the one-time detection of 20 plant growth hormones by combining the positive mode and the negative mode, without the need to prepare solutions and repeat the operation multiple times, which greatly reduces the time and reagent consumption and improves the work efficiency;

[0065] (3) The detection method provided by the present invention uses a particle size of 40μm-60μmC 18 Solid phase extraction adsorbent can achieve complete adsorption of growth hormone in strawberries, ensuring that the accuracy of test data is further improved during the detection process. DETAILED DESCRIPTION

[0066] The anhydrous magnesium sulfate used in the present invention needs to be processed in advance, burned at 550° C. for 4 hours, cooled to room temperature in a desiccator, and stored in a sealed bottle for later use.

[0067] The acetonitrile and formic acid used in the present invention are both of HPLC grade.

[0068] The instruments and equipment used in the present invention are as follows:

[0069] Liquid chromatography tandem mass spectrometer (Shimadzu LC-20AD; API4000)

[0070] Centrifuge

[0071] Analytical balance (accurate to 0.01g and 0.0001g)

[0072] Ultrasonic Cleaner

[0073] Rotary evaporator

[0074] Vortex mixer

[0075] Homogenizer

[0076] Centrifuge tube: 50mL, polypropylene (pp)

[0077] Microcentrifuge tube: 2mL.

[0078] Example

[0079] Example 1

[0080] A method for detecting 20 plant growth hormone residues in strawberries comprises the following steps:

[0081] (1) Preparation of standard products and standard solution:

[0082] Provide 20 kinds of plant growth hormone standards, specifically 2.4.-D, S-inducible hormone, 6-benzyladenine, paclobutrazol, chlorfenapyr, brassinolide, gibberellic acid, chlormequat, mepiquat, clopyralid, p-chlorophenoxyacetic acid, naphthylacetic acid, indoleacetic acid, thidiazuron, uniconazole, butyric acid hydrazide, trinexapac-ethyl, diethylaminoethyl, 2,3,5-triiodobenzoic acid, and ethephon;

[0083] Prepare the standard solution for later use: accurately weigh the above 20 kinds of standard substances, add them into volumetric flasks, dilute to the scale with acetonitrile, and prepare a standard solution with a concentration of 1000 μg / mL (20 bottles in total, stored at -18℃ in a dark place).

[0084] (2) Test solution configuration:

[0085] Weigh the strawberries, take the edible part, and homogenize it. Weigh 5.0g of the homogenized sample to be tested, place it in a 50mL centrifuge tube, add 20mL of acetonitrile solution containing 1% acetic acid (v / v), 4.0g of anhydrous magnesium sulfate, and 1.0g of anhydrous sodium acetate, homogenize at high speed for 5min, centrifuge at 400r / min for 5min, purify it after treatment to obtain the supernatant, take 5.0mL of the upper solution and add it to a 50mL centrifuge tube pre-added with 250mgC 18 and 750 mg of anhydrous magnesium sulfate in a centrifuge tube, vortex for 30 seconds, centrifuge at 4000 r / min for 5 minutes, take 0.5 mL of the supernatant into a 2 mL microcentrifuge tube, add 0.5 mL of 0.1% formic acid aqueous solution and mix well, filter through a 0.2 μm microporous membrane, and prepare the test solution for use;

[0086] It also includes the preparation of a mixed standard solution, and the method for preparing the mixed standard solution includes the following steps: taking the prepared standard solution, preparing a mixed standard solution of 20 plant growth hormones with a concentration of 1.0 μg / mL with acetonitrile, and storing it at 0°C to 4°C away from light.

[0087] The method also includes the preparation of a mixed standard working solution, wherein the method comprises the following steps: taking the mixed standard solution, preparing mixed standard working solutions of various concentrations with a blank sample matrix, and preparing and using the mixed standard working solutions immediately. The concentration range of the standard curve is 0.005 mg / L-0.1 mg / L.

[0088] (3) Test condition setting:

[0089] Setting positive mode liquid chromatography conditions and negative mode liquid chromatography conditions;

[0090] The positive mode liquid chromatography conditions include:

[0091] Mobile Phase:

[0092] Phase A: acetonitrile; Phase B: 0.1% formic acid aqueous solution

[0093] Flow rate: 0.2mL / min

[0094] Column temperature: 40°C

[0095] Injection volume: 10 μL

[0096] Chromatographic column: Agilent ZORBA×Eclipse Plus C18, 100×2.1mm, 3.5μm (or equivalent column) Analysis time: 14min

[0097] The mobile phase elution gradient is shown in Table 1 below

[0098] Table 1: Positive mode mobile phase elution gradient

[0099]

[0100] The negative mode liquid chromatography conditions include:

[0101] Mobile Phase:

[0102] Phase A: acetonitrile; Phase B: water

[0103] Flow rate: 0.3mL / min

[0104] Column temperature: 40°C

[0105] Injection volume: 10 μL

[0106] Chromatographic column: Agilent ZORBA×Eclipse Plus C18, 100×2.1mm, 3.5μm (or equivalent column) Analysis time: 14min

[0107] The mobile phase elution gradient is shown in Table 2 below

[0108] Table 2 Negative mode mobile phase elution gradient

[0109]

[0110]

[0111] In this application, the negative mode mass spectrometry parameters of 11 growth hormones are shown in Table 3.

[0112] Table 3: Negative mode mass spectrometry parameters of 11 plant growth hormones

[0113]

[0114]

[0115] Note: The ion pairs marked with * are quantitative ion pairs. In this application, the negative mode mass spectrometry parameters of 9 growth hormones are shown in Table 4.

[0116] Table 4: Positive mode mass spectrometry parameters of 9 plant growth hormones

[0117]

[0118] Note: The ion pairs marked with * are quantitative ion pairs.

[0119] Sample detection: The mixed standard working solution and the test solution are subjected to chromatographic detection.

[0120] The solutions used in the examples are as follows:

[0121] The acetonitrile solution containing 1% acetic acid is specifically prepared as follows: 10 mL of acetic acid (superior grade) is added to a 1000 mL volumetric flask, and then 990 mL of acetonitrile is added to the scale, shaken well, and set aside.

[0122] The specific method of 0.1% formic acid aqueous solution is as follows: 1.0 mL of formic acid (HPLC grade) is added into a 1000 mL volumetric flask, and then water is added to make up to the scale line, shaken and set aside.

[0123] Test Results

[0124] Judging criteria:

[0125] The retention time of the chromatographic peak of the target compound in the sample solution to be tested should be consistent with the retention time of the corresponding matrix spiked standard chromatographic peak, and the variation range should be within ±2.5%.

[0126] The mass spectrometry qualitative ions of each compound must appear, including at least one parent ion and two daughter ions. For the same batch of the same compound, the relative abundance ratio of the two daughter ions of the compound in the sample is compared with the standard solution of equivalent concentration. The allowable deviation of the relative abundance ratio should not exceed the range specified in Table 5.

[0127] Table 5 Maximum allowable deviation of relative ion abundance

[0128]

[0129] Calculation of the content of a certain plant growth hormone in the sample solution to be tested:

[0130]

[0131] Where: X——Content of a certain plant growth hormone in the sample (mg / kg)

[0132] C——Concentration of a plant growth hormone measured in the sample solution to be tested on the machine (μg / L)

[0133] V——Constant volume of the sample solution to be tested (mL)

[0134] M——mass of the sample solution to be tested (g)

[0135] DF——Dilution factor

[0136] The calculation result is accurate to one decimal place.

[0137] Accuracy (recovery rate): Calculated by the recovery rate of the spiked sample, taking the negative blank sample for 3 levels of recovery, 6 parallel tests, the specific preparation process is as follows:

[0138] Low concentration level spike: Accurately weigh 5g of sample, add 50μL of 1mg / L intermediate liquid of 20 mixed standards, and then test it on the machine according to the test requirements of normal sample pretreatment. Test the same sample in 6 parallel tests.

[0139] Medium concentration level spike: Accurately weigh 5g of sample, add 150μL of 1mg / L intermediate liquid of 20 mixed standards, and then test it on the machine according to the test requirements of normal sample pretreatment. Test the same sample in 6 parallel tests.

[0140] High concentration level spike: Accurately weigh 5g of sample, add 500μL of 1mg / L intermediate liquid of 20 mixed standards, and then test it on the machine according to the test requirements of normal sample pretreatment. Test the same sample in 6 parallel tests.

[0141] The recovery rates of the above samples meet the requirements of GB / T 27404-2008. The specific results are shown in Table 6.

[0142] Precision: It is calculated by the relative standard deviation of the spiked sample. Take the negative blank sample to conduct the recovery rate addition level experiment, and calculate the precision by calculating the relative standard deviation of the positive result of the spiked sample. The specific preparation process is as follows:

[0143] Low concentration level spike: Accurately weigh 5g of sample, add 50μL of 1mg / L intermediate liquid of 20 mixed standards, and then test it on the machine according to the test requirements of normal sample pretreatment. Test the same sample in 6 parallel tests.

[0144] Medium concentration level spike: Accurately weigh 5g of sample, add 150μL of 1mg / L intermediate liquid of 20 mixed standards, and then test it on the machine according to the test requirements of normal sample pretreatment. Test the same sample in 6 parallel tests.

[0145] High concentration level spike: Accurately weigh 5g of sample, add 500μL of 1mg / L intermediate liquid of 20 mixed standards, and then test it on the machine according to the test requirements of normal sample pretreatment. Test the same sample in 6 parallel tests.

[0146] The precision of the above samples meets the requirement of RSD≤10%. The specific results are shown in Table 6

[0147] Table 6: Test results

[0148]

[0149]

[0150] The detection limit was tested with reference to 3 times the signal-to-noise ratio, and the quantification limit was tested with reference to 10 times the signal-to-noise ratio. The specific results are shown in Table 7.

[0151] Table 7: Summary of detection limit and quantification limit results

[0152]

[0153]

[0154] According to the above tests, when the sample weight is 5g and the fixed volume is 1mL, the detection limits of 2.4.-D, S-abscisic acid, 6-benzyladenine, paclobutrazol, chlorpyrifos, brassinolide, gibberellic acid, chlormequat, mepiquat, clopyralid, 4-chlorophenoxyacetic acid (abscisic acid), naphthylacetic acid, indoleacetic acid, thiadiol, uniconazole, butyric acid hydrazide, trinexapac-ethyl, ampicillin, 2,3,5-triiodobenzoic acid, and ethephon are all 0.05mg / kg, and the quantification limit is 0.1mg / kg. It has high accuracy, good sensitivity, and less matrix interference, and can be used as an effective method for screening whether to spray plant growth hormones.

Claims

1. A method for detecting 20 plant growth hormone residues in strawberries, characterized in that: The following steps are involved: Preparation of standard products and preparation of standard solutions: provide plant growth hormone standards and prepare the standards into standard solutions for standby use; Preparation of the test solution: Weigh the strawberries, take the edible part, homogenize it, purify it after treatment to obtain the supernatant, and prepare it as the test solution for later use; Test condition setting: setting positive mode liquid chromatography conditions and negative mode liquid chromatography conditions; Sample testing: The standard solution and the test solution are subjected to chromatographic testing.

2. The method for detecting 20 plant growth hormone residues in strawberries according to claim 1, characterized in that: The step of obtaining the supernatant specifically includes: weighing the homogenized sample to be tested, adding a dissolving solution and a solid adsorbent, homogenizing, centrifuging, and then taking the upper solution, adding a solid phase extraction adsorbent, and centrifuging to obtain a supernatant.

3. The method for detecting 20 plant growth hormone residues in strawberries according to claim 2, characterized in that: The solid adsorbent includes at least one of anhydrous magnesium sulfate, anhydrous sodium acetate, anhydrous calcium chloride and soda lime.

4. The method for detecting 20 plant growth hormone residues in strawberries according to claim 3, characterized in that: The solid adsorbent comprises anhydrous magnesium sulfate and anhydrous sodium acetate, and the weight ratio of the anhydrous magnesium sulfate to the anhydrous sodium acetate is (1-5):

1.

5. The method for detecting 20 plant growth hormone residues in strawberries according to claim 2, characterized in that: The solid phase extraction adsorbent is C 18 Solid Phase Extraction Adsorbents.

6. The method for detecting 20 plant growth hormone residues in strawberries according to claim 5, characterized in that: The C 18 The particle size of the solid phase extraction adsorbent is 40 to 60 μm.

7. The method for detecting 20 plant growth hormone residues in strawberries according to claim 1, characterized in that: The positive mode liquid chromatography conditions include: Mobile Phase: Phase A: acetonitrile; Phase B: 0.1% formic acid aqueous solution; Flow rate: 0.1~0.5mL / min; Column temperature: 40°C; Injection volume: 10 μL; Chromatographic column: Agilent ZORBA×Eclipse Plus C18, 100×2.1 mm, 3.5 μm; Analysis time: 10 to 15 minutes.

8. The method for detecting 20 plant growth hormone residues in strawberries according to claim 1, characterized in that: The negative mode liquid chromatography conditions include: Mobile Phase: Phase A: acetonitrile; Phase B: water; Flow rate: 0.1~0.5mL / min; Column temperature: 40°C; Injection volume: 10 μL; Chromatographic column: Agilent ZORBA×Eclipse Plus C18, 100×2.1 mm, 3.5 μm; Analysis time: 10 to 15 minutes.

9. The method for detecting 20 plant growth hormone residues in strawberries according to any one of claims 1 to 8, characterized in that: The 20 plant growth hormones include 2.4.-D, S-inducing hormone, 6-benzyladenine, paclobutrazol, chlormequat, brassinolide, gibberellic acid, chlormequat, mepiquat, clopyralid, p-chlorophenoxyacetic acid, naphthylacetic acid, indoleacetic acid, thidiazuron, uniclobutrazol, butyric acid hydrazide, trinexapac-ethyl, ampicillin, 2,3,5-triiodobenzoic acid, and ethephon.

10. Application of a method for detecting 20 plant growth hormone residues in strawberries, characterized in that: Used for the detection of growth hormone in fruits and vegetables.

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