Method for detecting residual quantity of glucocorticoid in organic fertilizer

Abstract

A method for detecting the residual quantity of glucocorticoid in an organic fertilizer comprises the following steps: 1, extraction: accurately weighing a sample in a centrifuge tube, adding a disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution, vigorously oscillating for 1 min, performing ultrasonic extraction and centrifugation at the speed of 5 / min, taking supernate and putting the supernate in another centrifuge tube, and collecting the supernate; adding the disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution into the residues again, repeatedly extracting twice, and fixing the volume to 50mL; b, purification: sucking supernate of the solution extracted in the step 1 into a centrifugal tube, and adding MgSO4, PSA and GCB into the centrifugal tube; carrying out vortex uniform mixing for 1 min, carrying out centrifugation for 5 min, and sucking the supernatant to pass through a microfiltration membrane; c, determining; and d, calculating to obtain the glucocorticoid content omega in the fertilizer. According to the method, efficient extraction and purification of the two glucocorticoids in the fertilizer are achieved, rapid and efficient detection of the glucocorticoids in the organic fertilizer is achieved, the detection time is short, the sensitivity is high, and the specificity is good.

Description

Technical Field

[0001] The invention belongs to the technical field of organic fertilizer detection, and particularly relates to a method for detecting glucocorticoid residues in organic fertilizer. Background Art

[0002] Glucocorticoids (GCS), also known as "adrenal cortex hormones," have the physiological function of regulating the metabolism of the three major substances in the human body (sugar, protein, and fat). They can also regulate the metabolism of potassium, sodium, and water to maintain the balance of the body's internal and external environment. They are the most important regulatory hormones for the body's stress response and are also the most commonly used and effective anti-inflammatory and immunosuppressant in clinical practice. Glucocorticoid drugs have important structural features required for biological activity, such as the δ-4,3-keto-11β,17α,21-trihydroxy structure contained in all natural and synthetic glucocorticoids. Common glucocorticoid drugs are mainly dexamethasone and betamethasone, which are Class II drugs and are all isomers. Long-term use may cause adverse reactions, such as reduced immunity, metabolic disorders and developmental abnormalities, or potential carcinogenic and teratogenic risks.

[0003] Currently, glucocorticoids are commonly used in livestock to treat inflammatory reactions, immune diseases, and ketosis in cattle. They are commonly used in production. Illegal uses of glucocorticoids are to increase feed intake in livestock, thereby increasing their weight. Consequently, countries around the world have established maximum residue limits for various hormones in animal-derived foods.

[0004] Organic fertilizer is the main means of production in my country's agricultural production. It plays a huge role in promoting food production and has a high contribution rate to food production. In particular, with the rapid development of green pesticides in recent years, organic fertilizer has become more and more popular. The widespread use of organic fertilizer is not only an important measure to improve agricultural productivity, but also an important role in improving the quality of farmland and maintaining ecological safety. Organic fertilizer is mostly processed from livestock manure. Veterinary drug residues that cannot be metabolized and absorbed in livestock will be excreted with the manure, and with the use of organic fertilizer, the agricultural production environment and agricultural products will be polluted again. However, since there is currently no effective method for detecting the amount of glucocorticoid drug residues in organic fertilizers, it is difficult to regulate the amount of glucocorticoid drug residues in organic fertilizers. Therefore, there is an urgent need for a method for detecting the amount of glucocorticoid drug residues in organic fertilizers in the prior art. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a method for detecting glucocorticoid residues in organic fertilizers.

[0006] The object of the present invention is achieved through the following technical scheme: a method for detecting glucocorticoid residues in organic fertilizer, comprising the following steps: a. Extraction: Accurately weigh the organic fertilizer sample in a 50mL centrifuge tube, add disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution, the mass volume ratio of the organic fertilizer sample to disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution is 0.2g / mL, shake vigorously for 1min, ultrasonically extract at 10r / min, centrifuge at a speed of not less than 5000r / min, centrifuge at 5 / min, take the supernatant and put it in another centrifuge tube, add the same amount of disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution as the first time to the residue, repeat the extraction twice, and make the volume to 50mL; b. Purification: The supernatant of the solution obtained in step 1 was transferred to a 10 mL centrifuge tube. A mixed purifier prepared by MgSO4, PSA, and GCB was added to the centrifuge tube. The mass volume ratio of the mixed purifier to the supernatant was 1 / 6 g / mL. In the mixed purifier, the mass ratio of MgSO4, PSA, and GCB was 60:10:1. Vortex mix for 1 min, centrifuge at 4000 rpm for 5 min, and aspirate the supernatant through a microporous filter membrane for determination. c. Determination: Liquid chromatography-tandem mass spectrometry was used under the following chromatographic conditions: column: Phenomenex Kinetex F5 (50×3.0 mm, 2.6 μm); mobile phase A: 0.1% formic acid in water with ammonium acetate; mobile phase B: acetonitrile; flow rate: 0.40 μL / min; column temperature: 40°C; detection wavelength: 355 nm; injection volume: 1.0 μl. d. Calculation of results: According to the quantitative ion peak area of ​​the glucocorticoid residue in the sample solution to be tested, combined with the matrix standard curve, the concentration ρ of the glucocorticoid in the sample solution to be tested is calculated, and the content ω of the glucocorticoid in the fertilizer is calculated according to the following formula: ω=ρ×V / m Where, ω is the amount of the analyte in the sample, in mg / kg; ρ is the mass concentration of the analyte in the sample solution obtained from the matrix-matched standard working curve, in ug / mL; V is the constant volume of the sample in mL; and m is the mass of the sample in g.

[0007] The disodium hydrogen phosphate citric acid-acetic acid acetonitrile solution is prepared by the following steps: a) Prepare 0.2 mol / L sodium dihydrogen phosphate solution: weigh 71.63 g of disodium dihydrogen phosphate, dissolve in water, and dilute to 1000 mL; b) Prepare a 0.2 mol / L aqueous solution of citric acid: weigh 42.03 g of citric acid and dilute to 1000 mL with water. c) Mix 0.2 mol / L sodium dihydrogen phosphate solution, 0.2 mol / L citric acid aqueous solution, and acetonitrile containing 0.1% acetic acid (containing 0.1% by volume of acetic acid and acetonitrile) in a volume ratio of 1:1:2 to obtain a sodium dihydrogen phosphate-citric acid-acetic acid-acetonitrile solution.

[0008] The method further comprises step e: performing calibration using a UPLC-MS / MS matrix external standard calibration curve method.

[0009] The present invention utilizes a liquid chromatography-tandem mass spectrometer, which primarily consists of a high-performance liquid chromatography (HPLC) section and a tandem mass spectrometer. The HPLC section is primarily used for sample separation and purification, while the tandem mass spectrometer section is used for sample detection and analysis. The HPLC-tandem mass spectrometer operates by pre-treating the sample before entering the HPLC section for separation and purification. The separated components then sequentially enter the mass spectrometer section, where they are first ionized into ions by an ion source and then enter a mass analyzer for mass analysis. The mass analyzer separates the ions according to their mass / charge ratio and sends the separated ions to a detector for detection and counting. The recorded mass spectra and chromatograms allow for further analysis and identification of the sample.

[0010] In the present invention, ultrasonic extraction using a sodium hydrogen phosphate, citric acid, and acetic acid acetonitrile solution improves extraction efficiency. Purification using a purifier obtained by using different reagent component ratios yields excellent purification effects and simple, convenient purification methods. Compared with traditional solid-phase extraction techniques, this method significantly improves timeliness and work efficiency, making it suitable for large-scale sample testing. Sample calibration using a matrix external standard curve method eliminates matrix effects.

[0011] The beneficial effects of the present invention are as follows: the present invention is a method for detecting glucocorticoids in organic fertilizer based on liquid chromatography-tandem mass spectrometry. Through an original organic fertilizer pretreatment step, the present invention achieves efficient extraction and purification of two glucocorticoids, dexamethasone and betamethasone, in fertilizer. At the same time, the present invention optimizes various operating parameters of the liquid chromatography-tandem mass spectrometry according to detection requirements, achieving rapid and efficient detection of glucocorticoids in organic fertilizer. The present invention has a short detection time, high sensitivity, and good specificity. It not only completes the detection process efficiently and accurately, but also reduces the amount of organic reagents used, is safe and environmentally friendly, and can better meet detection requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a bar graph of the extraction efficiency of different extraction solutions; Figure 2 These are the spectra of two glucocorticoid standards. The upper figure is the spectra of betamethasone, and the lower figure is the spectra of dexamethasone. Figure 3 This is a diagram showing the purification effect of three purifiers with different component ratios on the sample. DETAILED DESCRIPTION

[0013] The present invention is described in detail below with reference to the accompanying drawings. Example

[0014] 1. Instrument parameter optimization 1. Analytical and Instrumental Conditions: This method uses liquid chromatography-tandem mass spectrometry to detect glucocorticoid residues in organic fertilizers. Because glucocorticoid antibiotics contain multiple amino groups, are chemically unstable, and highly polar, the same chromatographic column type, mobile phase ratio, and gradient elution procedure were selected. Testing of target compound standards ultimately led to the selection of a Phenomenex Kinetex F5 (2.6 μm, 50 × 30 mm) column due to its high retention and efficiency. The study compared the separation effects of 0.1% acetic acid-methanol and 0.1% acetic acid-acetonitrile as mobile phases. The comparison found that the peak elution time of the 0.1% acetic acid-methanol mobile phase was relatively slow, and the response intensity was slightly higher than that of acetonitrile, but the response to dexamethasone was poor. In contrast, under the same flow rate conditions, the two analytes, dexamethasone and betamethasone, both eluted in the 0.1% acetic acid-acetonitrile mobile phase, and the peak elution time was earlier than that of the methanol mobile phase, and both components achieved maximum separation. Therefore, 0.1% acetic acid-acetonitrile was selected as the organic phase, and ammonium acetate was added to the aqueous phase of the mobile phase to significantly improve the sensitivity and signal-to-noise ratio.

[0015] Dexamethasone and betamethasone were eluted using gradient elution using 0.1% formic acid / ammonium acetate (containing 0.1% formic acid, 2 mmol / L ammonium acetate, and water) followed by 0.1% acetic acid / acetonitrile (containing 0.1% acetic acid and acetonitrile), and then 0.1% formic acid / water (containing 0.1% formic acid and water) followed by 0.1% acetic acid / acetonitrile. After validating various mobile phase combinations, the mobile phase gradient elution conditions shown in Table 1 were ultimately selected, using 0.1% formic acid / ammonium acetate and 0.1% acetic acid / acetonitrile as the mobile phase. Both dexamethasone and betamethasone were well retained during gradient elution, with good peak shapes and the absence of interfering peaks.

[0016] Table 1 Mobile phase gradient elution conditions time Mobile phase ratio A% (0.1% formic acid water ammonium acetate) Mobile phase proportion B% (0.1% acetic acid acetonitrile) Flow rate (uL / min) 0.00 95 5 0.40 2.00 95 5 0.40 6.00 5 95 0.40 6.01 5 95 0.40 8.00 95 5 0.40 10.00 95 5 0.40 2. Selection of Mass Spectrometry Conditions: Glucocorticoid antibiotics readily combine with one or more H+ species in SCAN mode to produce positively charged singly or multiply charged ions. The [M+H]+ ion peak response intensity is higher in positive ion mode. Therefore, a primary mass spectrometry scan was performed on the analytes dexamethasone and betamethasone to obtain parent ions, identify fragment ions, and then optimize the collision energy (CE) values ​​corresponding to the fragment ions. For dexamethasone, a parent ion of 393.2 was obtained in SCAN mode, and its fragment ions were 355 and 118. The declustering potential (DP) value corresponding to the fragment ion 355 was optimized. Comparison revealed that the corresponding abundances for collision energy (DP) values ​​of 30, 35, 50, and 73 eV were 5000, 5100, 5300, and 6000, respectively. Based on the highest response abundance, a declustering potential (CE) value of 73 eV was selected for the fragment ions 355 and 118. Betamethasone was scanned in SCAN mode to obtain a parent ion of 393.1, with fragment ions 373 and 168. Optimization of the declustering voltage (DP) values ​​corresponding to the fragment ions 373 and 168 revealed that the abundances corresponding to collision energy DP values ​​of 35, 40, 50, and 65 eV were 5000, 5300, 5100, and 5600, respectively. Therefore, a declustering voltage of 65 eV was selected for the fragment ions 373 and 168, and the ion map of the target was obtained after optimization. Given the target peak time of approximately 3.15 min, the mass spectrometer was set to switch to waste at 1-2.5 min and 3.5-10 min, with sample injection and detection at 2.5-3.5 min. This effectively maintains the instrument and protects the mass spectrometer end, extending its use time.

[0017] LC-MS / MS multiple reaction monitoring analysis was used to establish a selected ion database for multiple reaction monitoring (MRM) mode. Qualitative analysis was performed using retention time and parent ion / daughter ion pairs, while quantification was performed using peak area. In positive ESI scanning mode, the analytes were subjected to both a primary mass spectrometry full scan and a secondary mass spectrometry scan, acquiring specific MRM data. Each parameter was appropriately adjusted to maximize the signal and determine optimal mass spectrometry parameters. This experiment employed positive ESI (+) mode; detection was performed using multiple reaction monitoring (MRM), with qualitative analysis utilizing retention time and fragment signal ratios. The mass spectrometry conditions were as follows: ion spray voltage (IS): 5500 V, ion source temperature (TEM): 550°C; curtain gas pressure (CUR): 20 psi; collision gas (CAD): 9 mL / min; nebulizer gas pressure (Gas1): 30 psi; auxiliary drying gas (Gas2): 55 psi; entrance voltage (EP) = 10 V; dwell time (Dwell time): 50 ms. With these optimized conditions, glucocorticoid compounds were effectively separated, with short elution times and good peak shapes. Characteristic ions are shown in Table 2.

[0018] Table 2 Retention times and characteristic ions of two glucocorticoids

[0019] Through these experiments, the liquid chromatograph's mobile phase type, elution time, and retention time parameters were established. The mass spectrometer parameters, voltage pressure, and collision energy conditions were also established. Based on these optimized instrument parameters, the sample extraction and cleanup reagents used in pretreatment were optimized.

[0020] 2. Optimization of pre-treatment conditions 1. Selection of Extraction Reagents: Use appropriate extraction reagents to extract the target component from the sample matrix into the target solvent based on the differences in solubility of the target component in different solvents. Since glucocorticoid antibiotics contain multiple amino groups and are polar compounds, polar organic solvents are preferred for extraction. Therefore, organic extraction reagents such as methanol, acetonitrile, and ethyl acetate can be used. The present invention is used to detect residual glucocorticoids in organic fertilizers. Organic fertilizers are composed of biomass, animal and plant residues, excrement, and biological waste. The matrix is ​​relatively complex and contains a large number of trace elements and rich organic nutrients. It is readily soluble in water. Available nutrients such as phosphorus and potassium often exist as insoluble salts or complexes. The phosphate (HPO₄²⁻) in disodium hydrogen phosphate can combine with metal ions (such as Ca⁺ and Mg⁺) to form soluble complexes, promoting the release of nutrients from the fertilizer and facilitating subsequent detection. The moderate ionic strength of the disodium hydrogen phosphate solution dissolves the target component while reducing excessive dissolution of interfering substances such as organic matter, ensuring the clarity of the extract and reducing background interference in subsequent detection. At the same time, the ionic strength and pH value of the dipotassium hydrogen phosphate solution can inhibit the excessive dissolution of interfering substances such as organic matter and humic acid in the fertilizer, avoid turbidity of the extract, reduce background interference in subsequent detection, and ensure data accuracy. In view of this, this experiment uses dipotassium hydrogen phosphate citric acid, disodium hydrogen phosphate citric acid and organic reagents for mixed extraction. Five extraction reagents, including methanol, acetonitrile, ethyl acetate, dipotassium hydrogen phosphate citric acid-acetonitrile solution, and disodium hydrogen phosphate citric acid-acetic acid acetonitrile solution, were selected for extraction efficiency tests. The extraction results are shown in Figure 1 and Tables 3-7.

[0021] Wherein, the dipotassium hydrogen phosphate citric acid-acetonitrile solution is prepared by the following steps: 1) Prepare 0.2 mol / L potassium hydrogen phosphate solution: weigh 27.22 g of potassium hydrogen phosphate and dissolve it in water to make up to 1000 mL. 2) Prepare 0.2 mol / L citric acid solution: Weigh 42.03 g of citric acid and dilute to 1000 mL with water. 3) Mix 0.2 mol / L dipotassium hydrogen phosphate solution, 0.2 mol / L citric acid aqueous solution, and acetonitrile in a volume ratio of 1:1:2 to obtain a dipotassium hydrogen phosphate citric acid-acetonitrile solution.

[0022] Disodium hydrogen phosphate citric acid-acetic acid acetonitrile solution was prepared by the following steps: 1) Prepare 0.2 mol / L sodium dihydrogen phosphate solution: weigh 71.63 g of disodium dihydrogen phosphate, dissolve in water, and dilute to 1000 mL. 2) Prepare 0.2 mol / L citric acid solution: Weigh 42.03 g of citric acid and dilute to 1000 mL with water. 3) Mix 0.2 mol / L sodium dihydrogen phosphate solution, 0.2 mol / L citric acid aqueous solution, and acetonitrile containing 0.1% acetic acid (containing 0.1% acetic acid and acetonitrile by volume) in a volume ratio of 1:1:2 to obtain a sodium dihydrogen phosphate citric acid-acetic acid acetonitrile solution.

[0023]

[0024]

[0025]

[0026]

[0027] Comparing the three pure organic reagents, acetonitrile, methanol, and ethyl acetate, ethyl acetate offers the highest extraction recovery and best solubility. However, when extracting complex matrices (such as organic fertilizer), the extract is turbid, which affects the recovery of dexamethasone. Methanol also offers a relatively low recovery and is therefore not a suitable choice. Given the target compound's high solubility in water, acetonitrile is a more suitable organic phase for extraction. A comparison of the extraction results using dipotassium hydrogen phosphate (Citrate / Acetonitrile) and disodium hydrogen phosphate (Citrate / Acetonitrile) solutions reveals that the dipotassium hydrogen phosphate (Citrate / Acetonitrile) solution produces a clear, less turbid sample, but exhibits a mediocre chromatographic peak shape. Disodium hydrogen phosphate (Citrate / Acetonitrile) solution, on the other hand, exhibits a superior chromatographic peak shape and facilitates protein precipitation, improving extraction efficiency. This high extraction efficiency and minimal matrix interference led to the choice of disodium hydrogen phosphate (Citrate / Acetonitrile) solution as the extraction solution for the target compound.

[0028] 2. Selection of Cleanup Conditions: To reduce matrix interference and ensure instrument stability, a pretreatment method with excellent cleanup performance is essential. Traditional solid-phase extraction pretreatment methods are complex and time-consuming. The QUEChERS method, however, offers the advantages of rapidity, simplicity, and low cost. It can simultaneously purify multiple compounds, uses minimal solvent, and offers excellent recoveries and accuracy, making it a widely used sample pretreatment method. The QUEChERS method offers a wide range of cleanup agents for exploration. By varying the raw material ratio of the cleanup agent, matrix effects can be effectively reduced without significant adsorption of the target compound. Cleanup experiments were conducted using cleanup agents with varying raw material ratios to achieve the optimal non-destructive cleanup method. The selected cleanup agents include MgSO₄, PSA, and graphitized carbon black (GCB). MgSO₄ primarily removes water and other non-polar interferences from the sample and is highly hygroscopic; PSA primarily removes citric acid co-extractables, fatty acids, pigments, and polar interferences; and GCB primarily removes pigments and is suitable for darkly colored samples. According to the matrix characteristics of this study, the impurities that need to be removed are mainly fiber, protein and water in the sample, so the purifiers containing MgSO4, PSA and GCB were selected. According to the different proportions of each type of substance, they were divided into three groups. The grouping is shown in Table 8. The samples were purified with the above three groups of purifiers, and the purification effect was determined based on the addition recovery. Figure 3 It can be seen that group A has the best purification effect.

[0029] Table 8 Grouping of different purifiers Group <![CDATA[MgSO4 (Unit: mg)]]> PSA (unit: mg) GCB (unit: mg) Group A 150 25 2.5 Group B 50 10 0.5 Group C 350 50 5.0 After the above experimental demonstration, this technical solution screened out the technical means with the best extraction and purification effects, and established a method for detecting glucocorticoids in organic fertilizers based on liquid chromatography-tandem mass spectrometry.

[0030] 3. Detection test of glucocorticoid residues in organic fertilizers 1. Extraction: Accurately weigh 2.0 g of organic fertilizer sample into a 50 mL centrifuge tube, add 10.00 mL of disodium hydrogen phosphate, citric acid water-acetic acid acetonitrile solution to the sample, shake vigorously for 1 minute, ultrasonically extract at 10 r / min, centrifuge at a speed of not less than 5000 r / min, centrifuge at 5 / min, take the supernatant and put it into another centrifuge tube, add 10.00 mL of disodium hydrogen phosphate, citric acid water-acetic acid acetonitrile solution to the residue again, repeat the extraction twice, and adjust the volume to 50 mL (V).

[0031] Wherein, the sodium hydrogen phosphate citric acid-acetic acid acetonitrile solution is prepared by the following steps: 1) Prepare 0.2 mol / L sodium dihydrogen phosphate solution: weigh 71.63 g of disodium dihydrogen phosphate, dissolve in water, and dilute to 1000 mL. 2) Prepare 0.2 mol / L citric acid solution: Weigh 42.03 g of citric acid and dilute to 1000 mL with water. 3) Mix 0.2 mol / L sodium dihydrogen phosphate solution, 0.2 mol / L citric acid aqueous solution, and acetonitrile containing 0.1% acetic acid (containing 0.1% acetic acid and acetonitrile by volume) in a volume ratio of 1:1:2 to obtain a sodium dihydrogen phosphate citric acid-acetic acid acetonitrile solution.

[0032] 2. Purification: Pipette the supernatant of the solution obtained in step 1 into a 10 mL centrifuge tube. Add a mixed purifier consisting of 150 mg MgSO4, 25 mg PSA, and 2.5 mg GCB to the centrifuge tube. The mass volume ratio of the mixed purifier to the supernatant is 1 / 6 g / mL. Vortex mix for 1 min, centrifuge at 4000 rpm for 5 min, and aspirate the supernatant through a microporous filter membrane for determination.

[0033] 3. Determination: Liquid chromatography-tandem mass spectrometry was used, and the chromatographic conditions were as follows: 1) Chromatographic column: Phenomenex Kinetex F5 (50×3.0 mm, 2.6 μm); 2) Mobile phase A: 0.1% formic acid in water with ammonium acetate; Mobile phase B: acetonitrile; 3) Flow rate: 0.40 uL / min; 5) Column temperature: 40°C; 6) Detection wavelength: 355nm; 7) Injection volume: 1.0 μl; 4. Calculation of results: The concentrations of dexamethasone and betamethasone in the sample solution to be tested were calculated based on the quantitative ion peak area of ​​the residual dexamethasone and betamethasone in the sample solution to be tested and combined with the matrix standard curve. ρ , and calculate the content of dexamethasone and betamethasone in fertilizer according to the following formula ω , the calculation formula is as follows: ω=ρ × V / m in, ω : The residual amount of the tested residue in the sample, in mg / kg; ρ : mass concentration of the analyte in the sample solution obtained from the matrix-matched standard working curve, in μg / mL; V: constant volume of the sample, in mL; m: Weigh the sample mass in g.

[0034] 5. Calibration: Use the UPLC-MS / MS matrix external standard calibration curve method for calibration. In addition to the target analytes, the sample also contains other matrices, such as organic matter in fertilizers, which will produce matrix effects. The matrix effect will cause the ionization efficiency of the target to change (in the mass spectrometer), the chromatographic retention behavior to change or the peak shape to tail, and the detection signal to be suppressed or enhanced, resulting in quantitative deviation. The matrix standard is a standard solution prepared by adding the standard to a blank matrix of the same type as the sample, and its matrix composition is consistent with the sample to be tested. By establishing a standard curve through the matrix standard, the behavior of the target in the real matrix can be simulated to offset the influence of the matrix on the detection signal. When detecting glucocorticoid residues in fertilizers, the protein, calcium, potassium and other elements in the fertilizer matrix may inhibit the mass spectrometry signal of the detection substance. The matrix standard prepared with a blank fertilizer matrix can correct this inhibition, thereby Figure 2 It can be seen that the matrix standard working solution prepared with the matrix has a clean background and less interference.

[0035] Figure 2 The blank sample has a low baseline and little interference. The linear equation, linear range, and correlation coefficient of glucocorticoids are shown in Table 9. In the range of 2-20 ug / L, the linear equation of dexamethasone is y=4511.3x-520.0, and the correlation coefficient is R 2 Greater than 0.99, more reliable; within the range of 2-20 ug / L, the linear equation of tamethasone is y=6741.2x-141.0, and the correlation coefficient R 2 Greater than 0.99, more reliable.

[0036] Table 9 Linear equations, linear ranges, and correlation coefficients of two glucocorticoids name Linear equations Linear range (ug / L) <![CDATA[R 2 ]]> Dexamethasone y=4511.3x-520.0 2-20 0.998 Betamethasone y=0741.2x-141.0 2-20 0.997 6. Recovery and precision of the method of the present invention: To one organic fertilizer sample, 2 μg / kg, 10 μg / kg, and 20 μg / kg of dexamethasone and betamethasone were added, respectively, based on the mass of the organic fertilizer sample. After thorough mixing, the samples were extracted and purified according to the sample preparation method. A spiked recovery test was performed, and the test was repeated 6 times. The results are shown in Table 10. It can be seen from the table that the method of the present invention has a high sensitivity for detecting these two glucocorticoids.

[0037]

[0038] 7. Influence of organic fertilizer matrix effects: Due to the presence of numerous interfering substances other than the analyte in actual samples, these substances may enhance or inhibit the analyte during measurement. Specifically, the matrix effects of the present method were examined by comparing the response values ​​of the matrix-added solution and the standard solution after sample extraction and purification. A ratio equal to or close to 1 indicates the absence of matrix effects; a ratio greater than 1 indicates that the test compound exhibits ion enhancement; and a ratio less than 1 indicates that the test compound exhibits ion suppression. As shown in Table 11, both glucocorticoids exhibit strong matrix suppression effects in the fertilizer matrix. In the actual testing process, a matrix-matched standard curve was used in the quantitative analysis to correct for the influence of matrix effects on the results.

[0039]

[0040] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting glucocorticoid residues in organic fertilizers, characterized in that The steps include: a. Extraction: Accurately weigh the organic fertilizer sample in a 50mL centrifuge tube, add disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution, the mass volume ratio of the organic fertilizer sample to disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution is 0.2g / mL, shake vigorously for 1min, ultrasonically extract at 10r / min, centrifuge at a speed of not less than 5000r / min, centrifuge at 5 / min, take the supernatant and put it in another centrifuge tube, add the same amount of disodium hydrogen phosphate citric acid water-acetic acid acetonitrile solution as the first time to the residue, repeat the extraction twice, and make the volume to 50mL; b. Purification: Pipette the supernatant of the solution obtained in step 1 into a 10 mL centrifuge tube, add a mixed purifier prepared by MgSO4, PSA, and GCB to the centrifuge tube, and the mass volume ratio of the mixed purifier to the supernatant is 1 / 6 g / mL. In the mixed purifier, the mass ratio of MgSO4, PSA, and GCB is 60:10:1; vortex mix for 1 min, centrifuge at 4000 rpm for 5 min, and aspirate the supernatant through a microporous filter membrane for determination; c. Determination: Liquid chromatography-tandem mass spectrometry was used under the following chromatographic conditions: chromatographic column: Phenomenex Kinetex F5 (50×3.0 mm, 2.6 μm); mobile phase A: 0.1% formic acid in water with ammonium acetate; mobile phase B: acetonitrile; flow rate: 0.40 μL / min; column temperature: 40°C; detection wavelength: 355 nm; injection volume: 1.0 μl; d. Calculation of results: According to the quantitative ion peak area of ​​the glucocorticoid residue in the sample solution to be tested, combined with the matrix standard curve, the concentration ρ of the glucocorticoid in the sample solution to be tested is calculated, and the content ω of the glucocorticoid in the fertilizer is calculated according to the following formula: ω=ρ×V / m Where, ω is the amount of the analyte in the sample, in mg / kg; ρ is the mass concentration of the analyte in the sample solution obtained from the matrix-matched standard working curve, in ug / mL; V is the constant volume of the sample in mL; and m is the mass of the sample in g.

2. the detection method of glucocorticoid residual amount in a kind of organic fertilizer according to claim 1, is characterized in that: The disodium hydrogen phosphate citric acid-acetic acid acetonitrile solution is prepared by the following steps: a) Prepare 0.2 mol / L sodium dihydrogen phosphate solution: weigh 71.63 g of disodium dihydrogen phosphate, dissolve in water, and dilute to 1000 mL; b) Prepare a 0.2 mol / L aqueous solution of citric acid: weigh 42.03 g of citric acid and dilute to 1000 mL with water. c) Mix 0.2 mol / L sodium dihydrogen phosphate solution, 0.2 mol / L citric acid aqueous solution, and acetonitrile containing 0.1% acetic acid (containing 0.1% by volume of acetic acid and acetonitrile) in a volume ratio of 1:1:2 to obtain a sodium dihydrogen phosphate-citric acid-acetic acid-acetonitrile solution.

3. the detection method of glucocorticoid residual amount in a kind of organic fertilizer according to claim 2, is characterized in that: The method further comprises step e: performing calibration using a UPLC-MS / MS matrix external standard calibration curve method.

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