KR102061604B1 - Analysis method of prohexadione calcium remaining in agricultural products - Google Patents

Abstract

The present invention provides an analytical method capable of analyzing the residual analysis of prohexadione-calcium simply and quickly and accurately.
Prohexadione-calcium residual analysis method of the present invention comprises the steps of a) dissolving 10 mg of prohexadion-calcium (Prohexadion-calcium) in 0.1L of distilled water to prepare a concentration of 100mg / L; b) weighing and preparing 1 to 20 g of homogenized analytical sample of fruit, vegetables, or crops for analysis in a 50 mL centrifuge tube; c) extracting a residual component of prohexadione-calcium by adding 1-20 mL of acetonitrile containing 0.5-1% formic acid to the assay sample and shaking for 1-3 minutes; d) centrifuging for 1-10 minutes at 3,000-5,000 rpm / min using a centrifuge; e) taking 400 µl of the extracted supernatant, mixing with 600 µl of distilled water, adjusting the concentration to 1000 µl, and then using a 0.2 µm syringe filter to use as an assay solution; f) analysis by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

Description

Calcium remaining in agricultural products and analysis method thereof {Analysis method of prohexadione calcium remaining in agricultural products}

The present invention relates to a method for analyzing the residual amount of prohexadione-calcium using acetonitrile containing formic acid, and more particularly, to a method for analyzing the residual amount of prohexadione-calcium used as a growth inhibitor of fruit or crops. It is about.

Fruits are too early to be fruitful when nutrition is too vigorous, and excessive nutrient growth in trees also causes dense obstructions, making the inside of the water pipe complex and shading, resulting in poor pest control efficiency. Providing a way to maintain or improve flowering, fruiting, yield, fruit quality, etc. while reducing the size of water bodies is an important goal in fruit growing. Plant growth regulators are used for the purpose of artificially controlling the growth of crops to increase added value or increase yields, and auxins, gibberellins, cytokinins and the like are known.

Among the growth inhibitors used in the prior art, Ethephon (Ethephon) when sprayed at a concentration that can sufficiently inhibit the growth of eggplants, depending on the type or variety of fruit causes fruit fall, fruit softening, biennial side effects. Daminozide has the property of inhibiting the production and action of gibberellin, and has been used most successfully in apples to suppress the growth of eggplants, to enhance pigmentation and to improve the storage capacity of fruits, but it takes a relatively long time to decompose. It has been reported to be carcinogenic and has been banned for use in food crops, including apples. CCC (Chlorocholine chloride, Chlormequat, Cycocel) is a substance that inhibits gibberellin biosynthesis, and it has recently been deregistered in several countries due to its over-residual residue due to too frequent use. Paclobutrazol is also an inhibitor of Gibberellin's biosynthesis, which is particularly effective in inhibiting the growth of nuclear fruits such as cherry blossoms and peaches. Problems have arisen and are not used in Korea or abroad.

Therefore, it is well decomposed and does not affect fruit shape and quality without any residual, and inhibits the growth of fruit trees or crops according to the needs of plant growth regulators which can suppress the fruit growth and make the fruit fruit into a form suitable for cultivation and management. Prohexadione-Calcuim was developed as a plant growth regulator, and it is used as an inhibitor of aquatic growth by spraying on apple trees, but it does not affect the quality of crops such as fruits and the concentration of spray per unit area. There is a lack of research on this situation, and it is necessary to perform a rapid analysis of prohexadione-calcium remaining in the fruit for research and safety of agricultural products.

Conventional methods of prohexadione-calcium residue analysis include food processing or US EPA analysis.However, such analytical methods include pre-treatment methods such as `` extraction → suction filtration → decompression concentration → two times liquid distribution → decompression concentration → solid phase extraction. Solid Phase Extraction (SPE) cartridge purification → yongyong's complex process takes a lot of time, and in particular, complex pretreatment stages such as liquid distribution and condensation can reduce the recovery rate, resulting in poor accuracy of quantitative analysis. .

Korean Unexamined Patent Publication No. 10-1278631 B1 (2013. 06. 19)

In order to solve the above problems, the present invention provides an analytical method capable of analyzing the remaining components of prohexadione-calcium simply and quickly and accurately. The actual analyte is prohexadione, which is an active ingredient, and the present invention also uses acetonitrile containing formic acid when extracting prohexadione having the properties of a weakly acidic compound (pKa = 5.15) from analytical samples. Provided is an improved method for easily extracting residual components of hexadione-calcium.

In order to achieve the above object, the present invention provides a method for analyzing residual residue of prohexadione-calcium a) dissolving 10 mg of prohexadion-calcium in 0.1 L of tertiary distilled water to prepare a concentration of 100 mg / L. Making; b) weighing and preparing 1-20 g of homogenized analytical sample of fruit, vegetables, or crops for analysis in a 50 mL centrifuge tube; c) 1 to 20 mL of acetonitrile containing 0.5 to 1.0% formic acid was added to the analytical sample, followed by shaking at 1,000 to 10,000 rpm / min for 1 to 3 minutes, thereby remaining of prohexadione-calcium. Extracting the components; d) centrifuging at 3,000-5,000 rpm / min for 1-10 minutes using a centrifuge; e) taking 400 μl of the extracted supernatant, mixing 600 μl of distilled water, adjusting the amount to 1000 μl, and filtering it with a 0.2 μm syringe filter to use as an assay solution (unlike conventional crops in which the analytical sample contains moisture). In case of, weigh 1 ~ 5g of dry sample of fruit, fruit, vegetable or crop for analysis in 50mL centrifuge tube, mix with 5 ~ 10mL of water, and leave it for 1 ~ 2 hours to keep prohexadione-calcium. Used for component analysis); f) analysis by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

The present invention relates to a method for analyzing prohexadione-calcium in agricultural product samples, such as fruits and vegetables, provides a rapid pretreatment method within 10 minutes in the pretreatment process, a liquid chromatography-mass spectrometer (HPLC-MS / MS) It is effective to enable qualitative and quantitative analysis up to an undetectable level (0.01 ug / kg).

In addition, it is possible to analyze the residual components of the prohexadione-calcium to a simple and accurate low concentration than the conventional food deduction and the US EPA method provides a technically improved analysis method.

The present invention provides a method for analyzing residual components of Prohexadion-calsium, which is used for inhibiting or improving quality of fruit or crops.

In the pretreatment method used for the analysis, a standard material is prepared, an analytical sample is prepared, an analytical sample is extracted and distributed with acetonitrile, and then an analytical solution is prepared. Then analyze the instrument using a mass spectrometer. The present invention provides an improved method for analyzing residual components of prohexadion-calcium for use in inhibiting or improving the quality of fruit or crops.

In the pretreatment method used in the analysis, a standard material is prepared, an analytical sample is prepared, and then a sample of the agricultural product is extracted and purified with a reagent such as acetonitrile containing formic acid to prepare an assay solution. Then analyze the instrument using a mass spectrometer.

Prohexadion-calcium inhibits the fruit growth of fruit trees and does not adversely affect fruiting, yield and fruit quality, but also improves the quality and reduces the total quantity by about 30% due to the tree being dwarfed. It is a water growth inhibitor.

The prohexadion inhibits the growth by inhibiting the 3β-hydroxylation reaction from the GA ₂₀ intermediate to the final product GA ₁ during the Gibberellin biosynthesis process in the plant. Prohexadione-calcium also inhibits dioxigenase activity, which catalyzes many different reactions. In other words, prohexadione-calcium inhibits growth by inhibiting GAs-3-hydroxylase associated with 2-oxoglutarate and the enzymatic activity associated with it, thereby reducing GAs contributing to the renal kidney activity of fruit trees. In addition, it acts on ACC oxidase, which is related to ascorbic acid, reduces ethylene production, delays aging and inhibits falling fruit. In addition, flavanone 3-hydroxylae enzyme related to 2-oxaloglutarate is increased to increase flavonoids, a kind of phytochromosomes, and induce resistance to pathogens.

Prohexadione-calcium residual analysis from the agricultural products of the present invention is first prepared by dissolving 10 mg of prohexadion-calcium (Prohexadion-calcium) in 0.1L of distilled water to a concentration of 100mg / L to prepare a liquid standard.

  Next, weigh 1 ~ 20g of agricultural product analysis sample such as fruit, vegetables, and homogenized homogenizer as a sample for analysis in a 50mL centrifuge tube, and prepare aceto containing 0.5 ~ 1% formic acid in the sample. Add 1-20 mL of nitrile (Acetonitrile) and shake for 1 to 3 minutes at 1,000 to 10,000 rpm / min to extract prohexadione-calcium.

After extracting chlorohexadione-calcium, 1 ~ 8g MgSO ₄ and 0.5 ~ 2g NaCl are added and shaken for 1 ~ 3 minutes at 1,000 ~ 10,000rpm / min. After shaking as described above, the analysis sample containing formic acid is centrifuged at 3,000 to 5,000 rpm / min for 1 to 10 minutes using a centrifuge. Centrifugation is performed to separate the solid and the organic solvent layer. Then, 400 μl of the supernatant is mixed with 600 μl of distilled water, adjusted to 1000 μl, and filtered through a 0.2 μm syringe filter to prepare an assay solution for analysis.

  In particular, if the analytical sample is a dry sample from which moisture has been removed, rather than a normal crop sample containing water, 1 to 5 g of a dry sample of fruit, fruit, vegetable or crop for analysis in a 50 mL centrifuge tube is treated with water 5 After mixing with ~ 10mL and left for 1 to 2 hours to use the residual component analysis of prohexadione-calcium.

In order to extract the residual components of the prohexadione-calcium, acetonitrile containing 0.5-1% formic acid was added to the analytical sample and shaken. Then, 1-5 pieces of ceramic beads were added and 1-8g of MgSO ₄ and 0.5-2g After adding NaCl, shaking for 1 minute with a homogenizer is included. In the case of preparing a control sample (blank matrix), the same method as described above is used to prepare a calibration curve.

Purification of colored fruits and vegetables

Fruits and vegetables such as spinach, red paprika and carrots are high in nonpolar pigments such as carotenoids or chlorophyll. Some common matrix components, such as fatty acids and sugars, are efficiently removed, but samples that contain high levels of pigment require further processing. Graphitized carbon black (GCB) efficiently removes chlorophyll, a planar chlorophyll pigment, and can be further purified by adding graphitized carbon black, C18 and MgSO ₄ in the dispersive-SPE purification step.

(Example 1)

① Dissolve 10mg of prohexadione-calcium in 0.1L tertiary distilled water to prepare 100mg / L of standard substance.

② Weigh 10g (5g dry sample) of homogenized fruit or vegetable sample in 50mL centrifuge tube. Dry samples such as cereals and legumes are swelled by adding 10 mL of water and left for at least 1 hour.

③ After mixing 10 mL of acetonitrile (MeCN) containing 1% formic acid to the analytical sample, shake for 1 minute at 10,000 rpm / min.

④ Add 2 ceramic beads, add 4g of magnesium sulfate (MgSO ₄ ), 1g of sodium chloride (NaCl), and shake with a homogenizer for 10,000rpm / min for 1 minute.

⑤ After shaking in ④, centrifuge for 5 minutes at 3,000 rpm / min using a centrifuge.

⑥ Take 400 μl of the centrifuged supernatant, mix with 600 μl of distilled water, adjust to 1000 μl, filter with a syringe filter of 0.2 μm, and use it as a sample extract for analysis.

Standards and Sample liquid  Matrix matched calibration division Standard for preparing LC-MS / MS calibration curve (example) Sample liquid Cal. One Cal. 2 Cal. 3 Cal. 4 Concentration 0.5 μg / L 1 μg / L 2.5 μg / L 5 μg / L Preparation method Distilled water
500 μL Distilled water
500 μL Distilled water
500 μL Distilled water
500 μL

Distilled water
600 μL STD in distilled water
5 μg / L,
100 μL STD in distilled water
10 μg / L,
100 μL STD in distilled water
25 μg / L,
100 μL STD in distilled water
50 μg / L,
100 μL Blank
matrix
400 μL Blank
matrix
400 μL Blank
matrix
400 μL Blank
matrix
400 μL Sample Extract
400 μL

As shown in [Table 1], the target concentrations of the standard material for preparing the LC-MS / MS calibration curve are 5 µg / L, 10 µg / L, 25 µg / L, and 50 µg / L. The preparation method of the standard material was 400 μL of control matrix (Blank matrix) in 500 μg / L of distilled water, and the STD content was 50 μg / L, 100 μL (Cal. 1), 100 μL / L, and 100 μL (Cal. 2). , 250 µg / L concentration, 100 µL (Cal 3), 500 µg / L concentration, 100 µL (Cal. 4). The sample liquid is mixed with 600 μL of distilled water and 400 μL of sample extractant.

 LC-MS / MS Instrumental Analysis Conditions UPLC NASCA2 UPLC System, Nexera X2 Column Shiseido CAPCELL CORE C18 2.7μm, 2.1mmx150mm
Column Temperature: 40 ℃ Injector Injection volume: 10μL (adjust according to the sensitivity of the instrument)

Mobile
Phase



A: 0.1% formic acid in Water
B: 0.1% formic acid in Acetonitrile Time Flow (mL / min) A (%) B (%) 0 0.4 80 20 4 0.4 70 30 7 0.4 10 90 8.5 0.4 10 90 8.6 0.4 80 20 MS / MS Shimadzu 8050 Ionization Electrospray Ionization (ESI, negative) MS
Condition Interface Temperature: 150 ℃
DL Temperature: 250 ℃
Heat Block Temperature: 400 ℃
Interface Voltage: 4.0 kV
Conversion Dynode Voltage: 10.0 kV
Detector Voltage: 1.92 kV

It shows the measurement conditions of the liquid chromatograph and mass spectrometer using LC-MS / MS of [Table 2], the temperature of the column is 40 ℃, the injection of the injector is injected in an amount of 10μL, formic acid as a mobile phase solvent Using acetonitrile containing water and acetonitrile, the acetonitrile containing formic acid was flowed at a rate of 0.4 mL / min for 0 to 10 minutes with a composition of 20 to 90%. Insert the standard material and sample solution for calibration curve preparation into the Nexera X2 UPLC System mass spectrometer of [Table 2] and analyze the standard material and the sample.

 By ingredients MRM Multi-Reaction Monitoring condition ( Shimaezu  example) Ingredient Name R.T. Precursor
ion (m / z) Product
ion (m / z) CE Q1  Pre Bias (V) Q3  Pre Bias (V)
Prohexadione

4.62
210.90 167.05 26 13 13 210.90 123.20 25 11 14 210.90 111.10 23 20 21

Table 3 shows the monitoring results for each of the precursor ion and the product ion as MRM conditions of the mass spectrometer for prohexadione-calcium analysis. Precursor ion refers to the ionized original mass value of the target component, and product ion refers to the mass value at which the precursor ion is broken when given a constant energy. Collision energy (CE) is the constant energy applied to break the precursor ions. At this time, the retention time of prohexadione-calcium was 4.62 minutes. (Q1, Q3 Pre Bias (V) is the accelerating voltage given to Q1 and Q3, and it passes the selected ions quickly and losslessly.)

Chromatogram of Standard (10 μg / L in Apple sample)

<Prohexadione Calcium>

[Table 4] shows the results of analysis of the standard samples made for preparing the calibration curve through the process of ① ~ ⑥ apple control samples, liquid chromatograph-mass spectrometer, the prohexadione-calcium of 40 ㎍ / L The samples contained at low concentrations showed high selectivity and sensitivity.

A calibration curve is prepared by calculating the height or area of each peak on the chromatogram obtained by injecting the standard solution into the analyzer for each concentration. Qualitative confirmation of prohexadione-calcium requires that the retention time and measured mass value (m / z) of the test solution match the retention time and measured mass value (m / z) of the standard solution, and that the peak ratio of the ion pair (± 30% or less). In the quantitative confirmation, the concentration of the sample solution is calculated by substituting the measured value in the sample solution into the calibration curve, and the value of the concentration of prohexadione-calcium remaining in the sample is calculated by reflecting the dilution factor.

Detection concentration (µg / kg) = Calibration concentration (µg / L) x (Amount of extraction solvent (mL) / Sample amount (g)) x (Instrument analysis solution (mL) / Preparative sample amount (mL))

Validation results (high concentration 100㎍ / kg, low concentration 50㎍ / kg) item Linearity
( R ² ) High concentration
Recovery rate
Average
(n = 5) High concentration
Recovery rate
Standard
Deviation High concentration
opponent
Standard
Deviation Low concentration
Recovery rate
Average
(n = 5) Low concentration
Recovery rate
Standard
Deviation Low concentration
opponent
Standard
Deviation detection
Limit
(Μg / kg) dose
Limit
(Μg / kg) Apple 0.9994 97.4 1.3 1.4 96.3 0.45 0.47 0.54 1.63 ship 0.9989 95.7 0.7 0.8 97.8 0.84 0.85 1.00 3.01 Strawberry 0.9985 100.9 1.3 1.3 98.5 0.59 0.60 0.71 2.13 tomato 0.9972 93.2 1.0 1.1 94.0 0.68 0.72 0.81 2.44 paprika 1.0000 94.6 0.6 0.6 94.77 0.77 0.81 0.92 2.77

Table 5 shows the results of validating the vegetables or fruits. For apples, the quantitative limit was 1.63 ㎍ / kg, the detection limit was 0.54 ㎍ / kg, the pear was 3.01 ㎍ / kg, the detection limit was 1.00 ㎍ / kg, and the strawberry was 2.13 and the detection limit 0.71 ㎍ / kg, respectively. , Tomato had a quantitative limit of 2.44 ㎍ / kg, a detection limit of 0.81 ㎍ / kg, and paprika had a quantitative limit of 2.77 ㎍ / kg and a detection limit of 0.92 ㎍ / kg. The quantitative analysis of the tomato, strawberry, apple, pear, and paprika by quantitative calibration test showed that the linearity (R ² ) was more than 0.99. Within the range of 70-120% and relative standard deviation 20%, it is within the criteria for validation of the method development.

Claims (4)

In the method for analyzing the residual amount of prohexadione-calcium using HPLC-MS / MS instrumental analysis,
The column of the HPLC-MS / MS instrumental analysis is a non-polar column filled with octadesilane (C18) of 1.6 ~ 4μm in diameter, 50 ~ 150mm in length, 1 ~ 2.1mm in diameter, the column temperature is 35 ~ 40 ℃ Injector injection amount is 1 ~ 10uL
Composition A in the composition of the eluent used as the mobile phase in the HPLC-MS / MS instrumental analysis is water containing 0.1 wt% formic acid, composition B is acetonitrile containing 0.1 wt% formic acid, and the initial phase A composition of the mobile phase is 80% by weight, B composition is set to 20% by weight, the flow rate of the mobile phase is 0.2 to 0.4mL / min flowing for 1 to 10 minutes,
The method of analyzing the residual amount of prohexadione-calcium is mixed with 1 ~ 20g of analytical sample consisting of homogenized fruits or vegetables in a 50mL centrifuge tube, and added 1 ~ 20mL of acetonitrile containing 0.5 ~ 1% formic acid After shaking for 1 to 5 minutes at 1,000 to 10,000 rpm / min, and extracted prohexadione-calcium, 1 to 5 ceramic beads, 1 to 8 g of magnesium sulfate (MgSO4), 0.5 to 2 g of sodium chloride (NaCl) are added After shaking for 1 to 5 minutes at 1,000 to 10,000 rpm / min, centrifuging for 1 to 10 minutes at 3,000 to 5,000 rpm / min, taking 350 to 450 μl of supernatant and mixing it with 550 to 650 μl of distilled water. After filtration and use as an assay solution
If the analytical sample is a dry sample, prepared by weighing 1 ~ 5g of homogenized analytical sample of fruit, fruit or crop for analysis in a 50mL centrifuge tube and left for 1 to 2 hours by adding 5-10mL of water Residual amount analysis method of prohexadione-calcium characterized by using









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