Method for detecting 20 plant growth hormone residues in strawberry

Abstract

The application belongs to the technical field of detection methods, and more particularly relates to a method for detecting residues of 20 plant growth hormones in strawberries. The method specifically comprises the following steps: standard preparation and standard solution configuration: providing plant growth hormone standards, and configuring the standards into standard solutions for standby; test solution configuration: weighing strawberries, taking edible parts, homogenizing, purifying to obtain supernatant, and configuring into a test solution for standby; test condition setting: setting positive mode liquid chromatography conditions and negative mode liquid chromatography conditions; sample detection: performing chromatography detection on the standard solution and the test solution. The method provided by the application can simultaneously detect the 20 plant growth hormones present in strawberries, the accuracy of the detection method is reliable, the detection limit is 0.1 mg / kg, meets the detection requirements, and has the value of being popularized and applied.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection methods, and more particularly relates to a method for detecting the residual amounts of 20 plant growth hormones in strawberries. BACKGROUND

[0002] Strawberries are widely cultivated fruits, and their growth and development processes may be regulated by various plant growth hormones. These hormones include auxins, cytokinins, etc., which play a key role in the growth, flowering, fruiting, etc. of strawberries. However, in order to increase yield or improve quality, some growers may use exogenous hormones, such as expanders, to treat strawberries. The residues of these hormones may have adverse effects on human health, so it is particularly important to detect the growth hormones in strawberries.

[0003] Chinese patent CN103969363A discloses a method for detecting plant growth regulator residues in fruits. The disclosed patent records that by using the QuEChERS method, the plant growth regulator residues in fruits are extracted and purified by adding acetonitrile containing acetic acid, so that the standard addition recovery rate of the target compound reaches 71%-98%, the relative standard deviation is not higher than 6%, it has good sensitivity and selectivity, the recovery rate is stable, and UPLC-MS / MS is used to simultaneously detect forchlorfenuron, ethephon, 4-chlorophenoxyacetic acid sodium and 2,4-dichlorophenoxyacetic acid sodium, the method is simple, and the detection limits of the four target compounds reach 0.5-100.0 μg / kg.

[0004] However, the disclosed patent can only detect four growth hormones, which is not suitable for strawberries and other fruits containing multiple growth hormones on the market. Moreover, the detection method is complex, and the application value is not high. It is an important technical breakthrough to develop a method for simultaneously detecting the residual amounts of multiple growth hormones. SUMMARY

[0005] To solve the problems in the prior art, the first aspect of the present application provides a method for detecting the residual amounts of 20 plant growth hormones in strawberries, comprising the following steps:

[0006] Standard preparation and standard solution configuration: providing plant growth hormone standards, and configuring the standards into standard solutions for standby;

[0007] Test solution configuration: weighing strawberries, taking the edible part, homogenizing treatment, purifying the supernatant after treatment to obtain a test solution for standby;

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

[0009] Sample detection: performing chromatographic detection on the standard solution and the test solution.

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

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

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

[0013] In some preferred embodiments, 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.

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

[0015] In some preferred embodiments, the solid-phase extraction adsorbent is C 18 The solid-phase extraction adsorbent.

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

[0017] In some preferred embodiments, the specific steps of preparing the sample solution comprise: weighing the strawberries, taking the edible part, homogenizing, weighing the homogenized sample to be tested 5.00 g, placing it in a 50 mL centrifuge tube, adding 20 mL of acetonitrile solution containing 1% acetic acid (v / v), 4.0 g of anhydrous magnesium sulfate, and 1.0 g of anhydrous sodium acetate, homogenizing at high speed for 5 min, centrifuging at 400 r / min for 5 min; then taking 5.0 mL of the supernatant solution into a centrifuge tube to which 250 mg of C 18 and 750 mg of anhydrous magnesium sulfate have been added, vortexing for 30 s, centrifuging at 4000 r / min for 5 min, taking 0.5 mL of the supernatant solution into a 2 mL microcentrifuge tube, adding 0.5 mL of 0.1% formic acid aqueous solution, mixing well, filtering through a 0.2 μm microporous membrane, and obtaining the sample solution for use.

[0018] The solution used in the present application is specifically as follows:

[0019] The acetonitrile solution containing 1% acetic acid is specifically prepared as follows: taking 10 mL of acetic acid (extra pure) into a 1000 mL volumetric flask, then adding 990 mL of acetonitrile to make up to the calibration mark, shaking well, and preparing for use.

[0020] 0.1% formic acid in water is prepared by pipetting 1.0 mL of formic acid (HPLC grade) into a 1000 mL volumetric flask, then adding water to the mark and mixing well.

[0021] In some preferred embodiments, the plant growth hormone standard specifically includes 2.4-D, S-antagonist (abscisic acid), 6-benzyladenine, paclobutrazol, brassinolide, gibberellic acid, mepiquat chloride, mephenate, clopyralid, chlorophenoxyacetic acid (fluoronil), naphthaleneacetic acid, indoleacetic acid, thidiazuron, uniconazole, butyryl hydrazine, prohexadione, amaranth ester, 2,3,5-triiodobenzoic acid, and ethylene.

[0022] In some preferred embodiments, the method for preparing the standard solution includes the following steps: weighing the plant growth hormone standard, dissolving it in acetonitrile to prepare a standard solution with a concentration of 1000 μg / mL, and storing it in the dark at -18°C.

[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 prepared standard solution, preparing a mixed standard solution with a concentration of 1.0 μg / mL of 20 plant growth hormones with acetonitrile, and storing it in the dark at 0-4°C.

[0024] In some preferred embodiments, it also includes the preparation of a mixed standard working solution, and the method for preparing the mixed standard working solution includes the following steps: taking the mixed standard solution, preparing a mixed standard working solution with each concentration using a blank sample matrix, and using it immediately after preparation.

[0025] Note: The blank sample matrix here refers to an 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] A phase: acetonitrile; B phase: 0.1% formic acid in water

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

[0030] Column temperature: 40°C

[0031] Injection volume: 10 μL

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

[0033] Analysis time: 10-15 min

[0034] The flow phase elution gradient for the positive mode is shown in Table 1 below

[0035] Table 1: Flow phase elution gradient for positive mode

[0036]

[0037] The flow rate for the positive mode liquid chromatography conditions can be illustratively comprised of 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min.

[0038] The analysis time for the positive mode liquid chromatography conditions can be illustratively comprised of 10 min, 11 min, 12 min, 13 min, 14 min, 15 min.

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

[0040] Flow phase:

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

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

[0043] Column temperature: 40 °C

[0044] Injection volume: 10 μL

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

[0046] Analysis time: 10-15 min

[0047] The flow phase elution gradient for the negative mode is shown in Table 2 below

[0048] Table 2: Flow phase elution gradient for negative mode

[0049]

[0050] The flow rate for the negative mode liquid chromatography conditions can be illustratively comprised of 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min.

[0051] The analysis time for the negative mode liquid chromatography conditions can be illustratively comprised of 10 min, 11 min, 12 min, 13 min, 14 min, 15 min.

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

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

[0054]

[0055] Note: marked * is the quantitative ion pair Table 9, the negative mode mass spectrometry parameters of 11 plant growth hormones in the present application are shown in Table 4.

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

[0057]

[0058] Note: marked * is the quantitative ion pair Table 9, the negative mode mass spectrometry parameters of 11 plant growth hormones in the present application are shown in Table 4.

[0059] The method for detecting 20 plant growth hormones in strawberries provided by the present application can accurately detect the content of 20 plant growth hormones in strawberries by combining positive mode and negative mode, which is convenient and efficient, has high accuracy, and can detect 20 plant growth hormones at the same time, thereby reducing the time for multiple detections and being worth popularization and use.

[0060] The detection method provided in the present application uses C 18 The solid phase extraction adsorbent can completely adsorb the plant growth hormones in strawberries, thereby further improving the accuracy of detection data in the detection process.

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

[0062] Advantages

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

[0064] (2) The detection method provided by the present application can realize one-time detection of 20 plant growth hormones by combining positive mode and negative mode, without the need for multiple solution preparation and repeated operation, thereby greatly reducing the time and reagent consumption and improving the work efficiency;

[0065] (3) The detection method provided by the present application uses C 18 The solid phase extraction adsorbent can completely adsorb the plant growth hormones in strawberries, thereby further improving the accuracy of detection data in the detection process. DETAILED DESCRIPTION

[0066] The anhydrous magnesium sulfate used in the present application needs to be treated in advance, calcined at 550℃ for 4h, cooled to room temperature in a dryer, and stored in a sealed bottle for standby.

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

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

[0069] Liquid chromatograph 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 material (pp)

[0077] Microcentrifuge tube: 2mL.

[0078] Examples

[0079] Example 1

[0080] A method for detecting the residual amount of 20 plant growth hormones in strawberries, comprising the following steps:

[0081] (1) Standard preparation and standard solution preparation:

[0082] 20 plant growth hormone standards are provided, specifically 2.4.-D, S-antagonin, 6-benzyladenine, paclobutrazol, forchlorfenuron, brassinolide, gibberellic acid, chlormequat, mepiquat, clopyralid, chlormequat, naphthaleneacetic acid, indoleacetic acid, thidiazuron, uniconazole, butyrylhydrazide, trinexapac-ethyl, profenofel, 2,3,5-triiodobenzoic acid, and ethylene.

[0083] The standards are prepared into standard solutions for standby: accurately weigh the above-mentioned 20 standards respectively, add to a volumetric flask, and respectively dilute to the calibration mark with acetonitrile to prepare a standard solution with a concentration of 1000μg / mL (a total of 20 bottles, stored at -18℃ in the dark).

[0084] (2) Preparation of the solution to be tested:

[0085] Take strawberries, take the edible part, homogenate, take 5.0 g of the homogenized sample to be tested, place it in a 50 mL centrifuge tube, add 20 mL of 1% acetic acid in acetonitrile solution (v / v), 4.0 g of anhydrous magnesium sulfate, 1.0 g of anhydrous sodium acetate, homogenize at high speed for 5 min, centrifuge at 400 r / min for 5 min, purify the supernatant after treatment, take 5.0 mL of the upper solution to a centrifuge tube pre-added with 250 mg of C 18 and 750 mg of anhydrous magnesium sulfate, vortex for 30 s, centrifuge at 4000 r / min for 5 min, take 0.5 mL of the supernatant to 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 into a test solution for standby;

[0086] It also includes a mixed standard solution configuration method, which includes the following steps: taking the above prepared standard solution, preparing a mixed standard solution with 20 plant growth hormone concentrations of 1.0 μg / mL with acetonitrile, and storing at 0°C-4°C in the dark.

[0087] It also includes a mixed standard working solution configuration method, which includes the following steps: taking the above mixed standard solution, and preparing a mixed standard working solution of each concentration with a blank sample matrix, and using it immediately. The concentration range of the standard curve is 0.005 mg / L-0.1 mg / L.

[0088] (3) Test condition setting:

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

[0090] The positive mode liquid chromatography conditions include:

[0091] Mobile phase:

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

[0093] Flow rate: 0.2 mL / min

[0094] Column temperature: 40°C

[0095] Injection volume: 10 μL

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

[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: 0.3 mL / min

[0104] Column temperature: 40 °C

[0105] Injection volume: 10 μL

[0106] Chromatographic column: Agilent ZORBAX Eclipse Plus C18, 100 x 2.1 mm, 3.5 μm (or equivalent column)

[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 plant 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 marked * is the quantitative ion pair table In this application, the negative mode mass spectrometry parameters of 9 plant growth hormones are shown in Table 4.

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

[0117]

[0118] Note: The marked * is the quantitative ion pair table

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

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

[0121] The 1% acetic acid in acetonitrile solution is prepared as follows: take 10 mL of acetic acid (superior grade) into a 1000 mL volumetric flask, then add 990 mL of acetonitrile to the mark line, shake well, and prepare for use.

[0122] The 0.1% formic acid aqueous solution is prepared as follows: take 1.0 mL of formic acid (HPLC grade) into a 1000 mL volumetric flask, then add water to the mark line, shake well, and prepare for use.

[0123] Test results

[0124] Judgment criteria:

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

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

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

[0128]

[0129] The content of a certain plant growth hormone in the sample solution to be tested is calculated as follows:

[0130]

[0131] In the formula: X-the content of a certain plant growth hormone in the sample (mg / kg)

[0132] C-the concentration of a certain plant growth hormone measured in the sample solution to be tested (μg / L)

[0133] V-the constant volume of the sample solution to be tested (mL)

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

[0135] DF-the dilution factor

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

[0137] Accuracy (recovery rate): the recovery rate of the spiked sample is calculated, and the recovery rate of the negative blank sample is calculated at three addition levels, and six parallel tests are performed, and the specific preparation process is as follows:

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

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

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

[0141] The recovery rate of the above sample meets the requirements of GB / T 27404-2008, and the specific results are shown in Table 6

[0142] Precision: the relative standard deviation of the spiked sample is calculated, the negative blank sample is taken for recovery rate addition level experiment, the relative standard deviation of the positive result of the spiked sample is calculated to calculate the precision, and the specific preparation process is as follows:

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

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

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

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

[0147] Table 6: detection results

[0148]

[0149]

[0150] The detection limit was tested with reference to 3 times the signal-to-noise ratio, and the quantitative 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 quantitative limit results

[0152]

[0153]

[0154] According to the above detection, when the sample weight is 5 g and the constant volume is 1 mL, the detection limit of 2.4-D, S-antagonist (abscisic acid), 6-benzyladenine, paclobutrazol, forchlorfenuron, brassinolide, gibberellic acid, mepiquat, mepiquat chloride, clopyralid, p-chlorophenoxyacetic acid (dichlorophenoxyacetic acid), naphthaleneacetic acid, indoleacetic acid, thidiazuron, uniconazole, butyryl hydrazine, trinexapac-ethyl, amaranth ester, 2,3,5-triiodobenzoic acid, and ethylene is 0.05 mg / kg, and the quantitative limit is 0.1 mg / kg. The accuracy is high, the sensitivity is good, the matrix interference is small, and it can be used as an effective method for screening whether plant growth hormones are sprayed.

Claims

1. A method for detecting the residue levels of 20 plant growth hormones in strawberries, characterized in that, Includes the following steps: Preparation of Standards and Preparation of Standard Solutions: Plant growth hormone standards are provided, and standard solutions are prepared from the standards for later use. Preparation of test solution: Weigh strawberries, take the edible parts, homogenize them, purify them to obtain the supernatant, and prepare it as the test solution for later use; Test conditions settings: Set the positive mode liquid chromatography conditions and the negative mode liquid chromatography conditions; Sample testing: The standard solution and the test solution were subjected to chromatographic analysis; The normal mode liquid chromatography conditions include: mobile phase: A phase: acetonitrile; B phase: 0.1% formic acid aqueous solution; flow rate: 0.1~0.5 mL / min; column temperature: 40℃; injection volume: 10 μL; chromatographic column: Agilent ZORBA×Eclipse Plus C 18 100×2.1mm, 3.5 μm; Analysis time: 10~15min; The negative mode liquid chromatography conditions include: mobile phase: phase A: acetonitrile; phase B: water; Flow rate: 0.1~0.5 mL / min; Column temperature: 40℃; Injection volume: 10 μL; Column: Agilent ZORBA×Eclipse Plus C 18 100×2.1mm, 3.5 μm; Analysis time: 10~15min; The 20 plant growth hormones include 2,4-D, S-inducer, 6-benzyladenine, paclobutrazol, chlormequat chloride, brassinolide, gibberellic acid, chlormequat chloride, mepiquatidine, dichloropyridine acid, p-chlorophenoxyacetic acid, naphthaleneacetic acid, indoleacetic acid, thidiazuron, uniconazole, butyrylhydrazine, anti-loosening ester, aminoethyl ester, 2,3,5-triiodobenzoic acid, and ethephon. The specific negative mode mass spectrometry parameters of 11 plant growth hormones are as follows: The specific positive mode mass spectrometry parameters of the nine plant growth hormones are as follows: For each compound, the mass spectrometry qualitative ion must be present, including at least one parent ion and two daughter ions.

2. The method for detecting the residue levels of 20 plant growth hormones in strawberries according to claim 1, characterized in that, The steps for obtaining the supernatant specifically include: weighing the homogenized sample to be tested, adding the dissolving solution and solid adsorbent, homogenizing, centrifuging, then taking the upper layer solution, adding the solid phase extraction adsorbent, centrifuging, and obtaining the supernatant.

3. The method for detecting the residue levels of 20 plant growth hormones 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 quicklime.

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

1.

5. The method for detecting the residue levels of 20 plant growth hormones in strawberries according to claim 2, characterized in that, The solid-phase extraction adsorbent is C. 18 Solid-phase extraction adsorbent.

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

7. The application of a method for detecting the residue levels of 20 plant growth hormones in strawberries according to any one of claims 1 to 6, characterized in that, Used for the detection of growth hormones in fruits and vegetables.

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