A method for detecting brassinolide in soil

Abstract

The present invention discloses a method for detecting brassinolide in soil, belonging to the field of measuring or analyzing materials by means of measuring the chemical or physical properties of the measuring materials. The method includes sample pretreatment, extraction, derivation, and detection. The detection method of the present invention has high precision, and the RSD of precision determination is 0.87 - 1.57%; the recovery rate of the detection method of the present invention is stable at different spiked concentrations, and the recovery rate is 97.4 - 103.5%.

Description

Technical Field

[0001] The invention relates to a method for detecting brassinolide in soil, and belongs to the field of measuring or analyzing materials by means of measuring the chemical or physical properties of the materials. Background Art

[0002] Brassinolide is a new type of green and environmentally friendly plant growth regulator. It can promote the growth of vegetables, melons, fruits and other crops by soaking seeds with appropriate concentrations of brassinolide and spraying them on stems and leaves, improve quality, increase yield, make the color brighter, and make the leaves thicker. It can also advance the time of tea leaf picking, and make melons and fruits have higher sugar content, larger size, higher yield, and better storage resistance.

[0003] Plant growth regulators in the pesticide market are mainly composed of synthetic sodium nitrophenolate and brassinolide. In practical applications, naturally extracted brassinolide has the best quality and better comprehensive economic benefits, and is more popular and used by farmers. Regardless of which type of plant hormone it belongs to, it is harmless to humans and animals, and the normal dosage is very safe and effective.

[0004] In recent years, researchers have used GCMS or HPLC to detect brassinolide in plant samples, but there has been no report on the detection of brassinolide in soil. Soil is the base for plant growth and the material basis for human survival. Therefore, the quality of soil directly affects human survival and development. Therefore, the study of the determination of brassinolide in soil has a very positive significance for improving the environmental quality and production capacity of soil and protecting people's health.

[0005] Compared with brassinolide in plant samples, brassinolide in soil is more firmly bound to the soil and the extraction is unstable, resulting in lower measurement precision.

[0006] So far, no detection method for brassinolide in soil has been found. With the increasingly serious environmental pollution, establishing a detection method for brassinolide in soil and monitoring brassinolide in soil are issues that need to be urgently addressed. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects of the prior art, and achieve high precision detection of brassinolide in soil through specific pretreatment and extraction steps, and further detection.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for detecting brassinolide in soil, the method comprising sample pretreatment, extraction, derivatization and detection.

[0010] The following is a further improvement of the above technical solution:

[0011] The sample pretreatment includes grinding and baking;

[0012] The method of grinding is to mix the air-dried soil sample passing through a sieve hole of 0.8 - 1.5 mm and non-porous ceramic balls, add deionized water, heat up to 90 - 95 °C, control the speed at 450 - 500 rpm, carry out grinding for 60 - 70 min, keep the temperature at 80 - 90 °C, and blow dry with nitrogen until there is no deionized water to obtain the grinding material;

[0013] The particle size of the non-porous ceramic balls is 1.2 - 2.0 mm;

[0014] The mass ratio of the air-dried soil sample to the non-porous ceramic balls is 4:4.5 - 5.5;

[0015] The mass ratio of the deionized water to the air-dried soil sample is 4 - 6:3;

[0016] The method of baking is to place the grinding material in a vacuum oven, control the vacuum degree at 35 - 45 Pa, control the temperature at 150 - 160 °C, bake for 140 - 160 min, then adjust the vacuum degree to 15 - 25 Pa, adjust the temperature to 200 - 210 °C, bake for 40 - 60 min, cool, and separate the non-porous ceramic balls to obtain the pretreated sample;

[0017] The extraction includes primary extraction, secondary extraction, and mixing and drying;

[0018] The method of primary extraction is to accurately weigh 2 g of the pretreated sample (accurate to 0.01 g), place it in a 50 mL plastic centrifuge tube, add 8 - 12 ml of formic acid - methanol solution, vortex for 50 - 70 s, carry out ultrasonic treatment at a frequency of 30 - 50 kHz for 25 - 35 min, then control the rotation speed at 3800 - 4500 r / min for centrifugation for 4 - 6 min, and take the supernatant to obtain the primary extraction supernatant and primary extraction soil;

[0019] In the formic acid - methanol solution, the volume ratio of formic acid to methanol is 1:800 - 1200;

[0020] The method of secondary extraction is to take the primary extraction soil, place it in a 50 mL plastic centrifuge tube, add 8 - 12 ml of ethanol - chloroform solution, vortex for 80 - 100 s, carry out ultrasonic treatment at a frequency of 40 - 50 kHz for 12 - 17 min, then control the rotation speed at 5500 - 6500 r / min for centrifugation for 4.5 - 5.5 min, and take the supernatant to obtain the secondary extraction supernatant;

[0021] In the ethanol-chloroform solution, the volume ratio of ethanol to chloroform is 1:95 - 105;

[0022] The method of mixing and drying is as follows: Mix the supernatant of the first extraction and the supernatant of the second extraction, control the frequency at 35 - 45 kHz for ultrasonic treatment, with an ultrasonic time of 4.5 - 5.5 min, then perform nitrogen blowing until the volume is reduced to 8 - 12% of the original volume to obtain the dried extract;

[0023] The method of derivatization is as follows: Add 200 μL of 3-aminophenylboronic acid-acetonitrile solution to the dried extract, keep the water bath temperature at 75 °C, derivatize for 30 min, then perform nitrogen blowing until the volume is reduced to 50% of the original volume, make up the volume to 1 ml with methanol, and filter through a 0.22 μm organic phase microporous filter membrane to obtain the test solution;

[0024] In the 3-aminophenylboronic acid-acetonitrile solution, the volume ratio of 3-aminophenylboronic acid to acetonitrile is 1:1000.

[0025] The method of detection is as follows: Perform liquid chromatography determination on the test solution, and the liquid chromatography conditions are as follows:

[0026] The chromatographic column is Gemini C 18 150 mm * 4.6 mm (i.d.), with a particle size of 5 μm;

[0027] The column temperature is 40 °C;

[0028] The flow rate is 0.5 ml / min;

[0029] The injection volume is 5 μl;

[0030] Mobile phase A is acetonitrile, and mobile phase B is ultrapure water;

[0031] The elution condition is isocratic elution with mobile phase A and mobile phase B in a volume ratio of 90:10, and the elution time is 7 min.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The detection method of the present invention has high precision, and the RSD of precision determination is 0.87 - 1.57%;

[0034] The detection method of the present invention has stable recoveries at different spiked concentrations, and the recoveries are 97.4 - 103.5%. Detailed implementation manners

[0035] Example 1

[0036] (1) Sample pretreatment

[0037] a. Grinding

[0038] Mix the air-dried soil sample passing through a 1-mm sieve hole and non-porous ceramic balls, add deionized water, heat to 100 °C, control the speed at 450 rpm, carry out grinding for 70 min while maintaining the temperature at 85 °C, and blow dry with nitrogen until there is no deionized water left to obtain the grinding material;

[0039] The particle size of the non-porous ceramic balls is 1.5 mm;

[0040] The mass ratio of the air-dried soil sample to the non-porous ceramic balls is 4:5;

[0041] The mass ratio of the deionized water to the air-dried soil sample is 5:3;

[0042] b. Baking

[0043] Place the grinding material in a vacuum oven, control the vacuum degree at 40 Pa, control the temperature at 155 °C, bake for 150 min, then adjust the vacuum degree to 20 Pa, adjust the temperature to 205 °C, bake for 50 min, cool, and separate the non-porous ceramic balls to obtain the pretreated sample.

[0044] (2) Extraction

[0045] a. Primary extraction

[0046] Accurately weigh 2 g of the pretreated sample (accurate to 0.01 g), place it in a 50-mL plastic centrifuge tube, add 10 mL of formic acid-methanol solution, vortex for 60 s, carry out ultrasonic treatment at a frequency of 35 kHz for 30 min, then centrifuge at a speed of 4000 r / min for 5 min, and take the supernatant to obtain the primary extraction supernatant and the primary extraction soil;

[0047] In the formic acid-methanol solution, the volume ratio of formic acid to methanol is 1:1000;

[0048] b. Secondary extraction

[0049] Take the primary extraction soil, place it in a 50-mL plastic centrifuge tube, add 10 mL of ethanol-chloroform solution, vortex for 90 s, carry out ultrasonic treatment at a frequency of 45 kHz for 15 min, then centrifuge at a speed of 6000 r / min for 5 min, and take the supernatant to obtain the secondary extraction supernatant;

[0050] In the ethanol-chloroform solution, the volume ratio of ethanol to chloroform is 1:100;

[0051] c. Mixing and blowing dry

[0052] Mix the supernatant of the first extraction and the supernatant of the second extraction, control the frequency to 40 kHz for ultrasonic treatment for 5 minutes, perform nitrogen blowing, and dry it to 10% of the original volume to obtain the dried extraction solution;

[0053] (3)Derivatization

[0054] Add 200 μL of 3-aminophenylboronic acid-acetonitrile solution to the dried extraction solution, keep the water bath temperature at 75 °C, derivatize for 30 minutes, perform nitrogen blowing, and dry it to 50% of the original volume, then make up the volume to 1 ml with methanol, and filter through a 0.22 μm organic phase microporous filter membrane to obtain the test solution;

[0055] In the 3-aminophenylboronic acid-acetonitrile solution, the volume ratio of 3-aminophenylboronic acid to acetonitrile is 1:1000.

[0056] (4)Detection

[0057] Perform liquid chromatography determination on the test solution. The liquid chromatography conditions are as follows:

[0058] The chromatographic column is Gemini C 18 150 mm * 4.6 mm (i.d.), particle size 5 μm;

[0059] The column temperature is 40 °C;

[0060] The flow rate is 0.5 ml / min;

[0061] The injection volume is 5 μl;

[0062] Mobile phase A is acetonitrile, and mobile phase B is ultrapure water;

[0063] The elution condition is isocratic elution with mobile phase A and mobile phase B in a volume ratio of 90:10, and the elution time is 7 minutes.

[0064] Example 2

[0065] (1)Sample pretreatment

[0066] a. Grinding

[0067] Mix the air-dried soil sample passing through a 0.8 mm sieve hole and non-porous ceramic balls, add deionized water, heat up to 90 °C, control the speed at 500 rpm, perform grinding for 60 minutes, keep the temperature at 80 °C, and dry it with nitrogen until there is no deionized water to obtain the grinding material;

[0068] The particle size of the non-porous ceramic balls is 1.2 mm;

[0069] The mass ratio of the air-dried soil sample to the non-porous ceramic balls is 4:4.5;

[0070] The mass ratio of the deionized water to the air-dried soil sample is 4:3;

[0071] b. Baking

[0072] Place the abrasive in a vacuum oven, control the vacuum degree at 35 Pa, control the temperature at 160 °C, bake for 140 min, then adjust the vacuum degree to 15 Pa, adjust the temperature to 210 °C, bake for 40 min, cool, and separate the non-porous ceramic balls to obtain a pretreated sample.

[0073] (2) Extraction

[0074] a. Primary extraction

[0075] Accurately weigh 2 g of the pretreated sample (accurate to 0.01 g), place it in a 50 mL plastic centrifuge tube, add 8 mL of formic acid-methanol solution, vortex for 50 s, perform ultrasonic treatment at a frequency of 30 kHz for 35 min, then centrifuge at a rotation speed of 3800 r / min for 6 min, and take the supernatant to obtain the primary extraction supernatant and the primary extraction soil;

[0076] In the formic acid-methanol solution, the volume ratio of formic acid to methanol is 1:800;

[0077] b. Secondary extraction

[0078] Take the primary extraction soil, place it in a 50 mL plastic centrifuge tube, add 8 mL of ethanol-chloroform solution, vortex for 80 s, perform ultrasonic treatment at a frequency of 40 kHz for 17 min, then centrifuge at a rotation speed of 5500 r / min for 5.5 min, and take the supernatant to obtain the secondary extraction supernatant;

[0079] In the ethanol-chloroform solution, the volume ratio of ethanol to chloroform is 1:95;

[0080] c. Mixing and drying

[0081] Mix the primary extraction supernatant and the secondary extraction supernatant, perform ultrasonic treatment at a frequency of 35 kHz for 5.5 min, perform nitrogen blowing, and dry to 8% of the original volume to obtain a dried extraction solution;

[0082] (3) Derivatization

[0083] Add 200 μL of 3-aminophenylboronic acid-acetonitrile solution to the dried extraction solution, keep the water bath temperature at 75 °C, derivatize for 30 min, perform nitrogen blowing, and dry to 50% of the original volume, then make up the volume to 1 mL with methanol, and filter through a 0.22 μm organic phase microporous filter membrane to obtain a test solution;

[0084] In the 3-aminophenylboronic acid-acetonitrile solution, the volume ratio of 3-aminophenylboronic acid to acetonitrile is 1:1000.

[0085] (4)Detection

[0086] Perform liquid chromatography determination on the test solution. The liquid chromatography conditions are as follows:

[0087] The chromatographic column is Gemini C 18 150 mm * 4.6 mm (i.d.), particle size 5 μm;

[0088] The column temperature is 40 °C;

[0089] The flow rate is 0.5 ml / min;

[0090] The injection volume is 5 μl;

[0091] Mobile phase A is acetonitrile, and mobile phase B is ultrapure water;

[0092] The elution condition is isocratic elution with mobile phase A and mobile phase B in a volume ratio of 90:10, and the elution time is 7 min.

[0093] Example 3

[0094] (1)Sample pretreatment

[0095] a. Grinding

[0096] Mix the air-dried soil sample passing through a 1.5 mm sieve hole and non-porous ceramic balls, add deionized water, heat up to 95 °C, control the speed at 480 rpm, perform grinding for 65 min, keep the temperature at 90 °C, and blow dry with nitrogen until there is no deionized water to obtain the grinding material;

[0097] The particle size of the non-porous ceramic balls is 2.0 mm;

[0098] The mass ratio of the air-dried soil sample to the non-porous ceramic balls is 4:5.5;

[0099] The mass ratio of the deionized water to the air-dried soil sample is 6:3;

[0100] b. Baking

[0101] Place the grinding material in a vacuum oven, control the vacuum degree at 45 Pa, control the temperature at 150 °C, bake for 160 min, then adjust the vacuum degree to 25 Pa, adjust the temperature to 200 °C, bake for 60 min, cool, and separate the non-porous ceramic balls to obtain the pretreated sample.

[0102] (2)Extraction

[0103] a. Primary extraction

[0104] Accurately weigh 2 g of the pretreated sample (accurate to 0.01 g), place it in a 50 mL plastic centrifuge tube, add 12 mL of formic acid - methanol solution, vortex for 70 s, perform ultrasonic treatment at a frequency of 40 kHz for 25 min, then centrifuge at a rotation speed of 4500 r / min for 4 min, take the supernatant to obtain the first - extraction supernatant and the first - extraction soil;

[0105] In the formic acid - methanol solution, the volume ratio of formic acid to methanol is 1:1200;

[0106] b. Second - extraction

[0107] Take the first - extraction soil, place it in a 50 mL plastic centrifuge tube, add 12 mL of ethanol - chloroform solution, vortex for 100 s, perform ultrasonic treatment at a frequency of 50 kHz for 12 min, then centrifuge at a rotation speed of 6500 r / min for 4.5 min, take the supernatant to obtain the second - extraction supernatant;

[0108] In the ethanol - chloroform solution, the volume ratio of ethanol to chloroform is 1:105;

[0109] c. Mixing and drying

[0110] Mix the first - extraction supernatant and the second - extraction supernatant, perform ultrasonic treatment at a frequency of 45 kHz for 4.5 min, perform nitrogen blowing, and dry to 12% of the original volume to obtain the dried extraction solution;

[0111] (3)Derivatization

[0112] Add 200 μL of 3 - aminophenylboronic acid - acetonitrile solution to the dried extraction solution, keep the water - bath temperature at 75 °C for 30 min, perform nitrogen blowing, and dry to 50% of the original volume, then make up the volume to 1 mL with methanol, and filter through a 0.22 - μm organic - phase microporous filter membrane to obtain the test solution;

[0113] In the 3 - aminophenylboronic acid - acetonitrile solution, the volume ratio of 3 - aminophenylboronic acid to acetonitrile is 1:1000.

[0114] (4)Detection

[0115] Perform liquid - chromatography determination on the test solution, and the liquid - chromatography conditions are as follows:

[0116] The chromatographic column is Gemini C 18 150 mm * 4.6 mm (i.d.), particle size 5 μm;

[0117] The column temperature is 40 °C;

[0118] The flow rate is 0.5 mL / min;

[0119] The sample injection volume is 5 μl;

[0120] Mobile phase A is acetonitrile and mobile phase B is ultrapure water;

[0121] The elution condition is isocratic elution with mobile phase A and mobile phase B in a volume ratio of 90:10, and the elution time is 7 min.

[0122] Comparative Example 1

[0123] On the basis of Example 1, the sample pretreatment step was omitted, and the remaining steps were the same for detection.

[0124] Comparative Example 2

[0125] On the basis of Example 1, in the extraction step, the secondary extraction step was omitted. After the first extraction, the first extraction solution was ultrasonicated, the frequency was controlled at 40 kHz, ultrasonicated for 5 min, and then nitrogen blown until the volume was reduced to 10% of the original volume to obtain the dried extraction solution. The remaining steps were the same for detection.

[0126] Precision determination of Example 4

[0127] The test sample was the black loess soil reference material GBW07494 (HTSB-2). A sufficient amount of the soil composition analysis reference material was weighed and spiked. The spiked amount of brassinolide was 0.006 g / g. It was detected by the methods of Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2, respectively, and determined in parallel 6 times. The results were statistically calculated, and the determination results are shown in Table 1.

[0128] Blank spiked recovery determination of Example 5

[0129] The test sample was the black loess soil reference material GBW07494 (HTSB-2). A sufficient amount of the soil composition analysis reference material was weighed and divided into five groups, and spiked respectively. The spiked amounts of brassinolide were 0.002 g / g, 0.005 g / g, 0.0100 g / g, 0.025 g / g, and 0.050 g / g. It was detected by the methods of Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2, respectively. The content of brassinolide was determined and statistically calculated, and the spiked recovery rate was calculated. The determination results are shown in Table 2.

[0130]

[0131]

Claims

1. A method for detecting brassinolide in soil, characterized in that, the method includes sample pretreatment, extraction, derivatization, and detection; the sample pretreatment includes grinding and baking; the method of grinding is to mix the air-dried soil sample passing through a sieve hole of 0.8 - 1.5 mm and the non-porous ceramic balls, add deionized water, heat up to 90 - 95 °C, control the speed at 450 - 500 rpm, conduct grinding for 60 - 70 min, keep the temperature at 80 - 90 °C, and blow dry with nitrogen until there is no deionized water to obtain the grinding material; in the grinding step, the particle size of the non-porous ceramic balls is 1.2 - 2.0 mm, the mass ratio of the air-dried soil sample to the non-porous ceramic balls is 4:4.5 - 5.5, and the mass ratio of deionized water to the air-dried soil sample is 4 - 6:3; the method of baking is to place the grinding material in a vacuum oven, control the vacuum degree at 35 - 45 Pa, control the temperature at 150 - 160 °C, bake for 140 - 160 min, then adjust the vacuum degree to 15 - 25 Pa, adjust the temperature to 200 - 210 °C, bake for 40 - 60 min, cool, and separate the non-porous ceramic balls to obtain the pretreated sample; the extraction includes primary extraction, secondary extraction, and mixing and drying; the method of primary extraction is to accurately weigh 2 g of the pretreated sample, add 8 - 12 ml of formic acid - methanol solution, vortex for 50 - 70 s, control the frequency at 30 - 50 kHz for ultrasonic treatment for 25 - 35 min, then control the rotation speed at 3800 - 4500 r / min for centrifugation for 4 - 6 min, and take the supernatant to obtain the primary extraction supernatant and the primary extraction soil; in the formic acid - methanol solution, the volume ratio of formic acid to methanol is 1:800 - 1200; the method of secondary extraction is to take the primary extraction soil, add 8 - 12 ml of ethanol - chloroform solution, vortex for 80 - 100 s, control the frequency at 40 - 50 kHz for ultrasonic treatment for 12 - 17 min, then control the rotation speed at 5500 - 6500 r / min for centrifugation for 4.5 - 5.5 min, and take the supernatant to obtain the secondary extraction supernatant; in the ethanol - chloroform solution, the volume ratio of ethanol to chloroform is 1:95 - 105; the method of mixing and drying is to mix the primary extraction supernatant and the secondary extraction supernatant, control the frequency at 35 - 45 kHz for ultrasonic treatment for 4.5 - 5.5 min, and conduct nitrogen blowing to obtain the dried extraction solution; the method of derivatization is to add 200 μL of 3-aminophenylboronic acid - acetonitrile solution to the dried extraction solution, keep the water bath temperature at 75 °C, derivatize for 30 min, after nitrogen blowing, dilute to 1 ml with methanol, and filter through a 0.22 μm organic phase microporous filter membrane to obtain the test solution; in the 3-aminophenylboronic acid - acetonitrile solution, the volume ratio of 3-aminophenylboronic acid to acetonitrile is 1:1000; in the mixing and drying step, nitrogen blowing is carried out until the volume is 8 - 12% of the original; in the derivatization step, nitrogen blowing is carried out until the volume is 50% of the original; The detection method is to perform liquid chromatography on the test solution, and the liquid chromatography conditions are as follows: The chromatographic column is Gemini C18, 150 mm × 4.6 mm (i.d.), with a particle size of 5 μm; The column temperature is 40 °C; The flow rate is 0.5 ml / min; The injection volume is 5 μl; Mobile phase A is acetonitrile, and mobile phase B is ultrapure water; The elution condition is isocratic elution with mobile phase A and mobile phase B in a volume ratio of 90:10, and the elution time is 7 min.

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