Method for determining carvacol content in compound fertilizer

Abstract

The application discloses a method for determining the content of carvacol in compound fertilizer, and the method comprises gas phase determination and liquid chromatography, and the gas chromatography method for determining the content of carvacol in compound fertilizer comprises the following steps: S1, sample pretreatment; S2, matrix purification; S3, chromatographic analysis; and S4, quantitative calculation; in the application, a silica gel SPE column is used to replace a traditional diatomite column in the GC method, the polarity impurity adsorption is enhanced through ethyl acetate-hexane gradient activation, sodium chloride salting-out and EDTA complex metal ions are introduced in the HPLC method, the number of miscellaneous peaks in the GC chromatogram is reduced, the separation degree of the carvacol peak is improved, and the service life of the HPLC column is prolonged; the GC method is operated under the conditions of temperature control ultrasonic at 35±2 DEG C and full light avoidance; the HPLC method adopts room temperature methanol-water system extraction, and the recovery rate of the GC method is improved; the HPLC method avoids the 60 DEG C concentration requirement of acetonitrile extraction, and the light stability is improved.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection technology, specifically relating to a method for determining the content of carvacrol in compound fertilizer. Background Technology

[0002] In the field of agricultural fertilizer additive detection, monitoring the content of carvacrol as a natural antibacterial component is crucial. Currently, the mainstream methods are based on the detection standards for carvacrol in feed (such as GB / T36858-2018), using gas chromatography-mass spectrometry (GC-MS) or high performance liquid chromatography (HPLC). Conventional GC methods involve extracting feed samples with n-hexane, purifying them with a diatomaceous earth column, and then analyzing them. HPLC methods often use an acetonitrile-water system for direct extraction, followed by separation and ultraviolet detection using a C18 column.

[0003] However, the above methods have significant limitations when applied to complex matrices such as compound fertilizers. The matrix purification is incomplete, the diatomaceous earth column in the feed standard has insufficient adsorption capacity for high concentrations of phosphate and metal ions in fertilizers, impurity peaks seriously interfere with the target analytes during GC analysis (such as humic acid derivatives and carvacrol co-eluent on the HP-5 column), salt accumulation causes column blockage during HPLC detection, and the 0.45μm filter membrane cannot retain colloidal particles.

[0004] The loss of volatile components is due to the lack of temperature control design in the ultrasonic extraction process of fertilizers (current standard requires ≤60℃), while carvacrol volatilizes significantly above 40℃, especially in the nitrogen blowing concentration stage of GC method, where the loss rate is as high as 18%-32%, resulting in negative quantitative deviation.

[0005] Based on the above, we propose a method for determining the carvacrol content in compound fertilizers, which specifically addresses the aforementioned problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining the content of carvacrol in compound fertilizers and its preparation method. By reconstructing a dual-path detection system, the invention systematically solves the problem of quantifying carvacrol in fertilizers. The GC path, based on low-temperature ultrasonic extraction and silica gel column polarization purification, overcomes the bottleneck of recovery of thermally unstable molecules in complex matrices, enabling the detection accuracy of solid fertilizers (RSD≤3.5%) to reach the standard of pesticide residue analysis. The HPLC path relies on a methanol-water dynamic solvent system and C18 column synergistic purification to eliminate the masking effect of high-salt / high-humic acid matrices on ultraviolet detection, achieving accurate quantification within a wide linear range of 10-1000 mg / kg.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for determining the carvacrol content in compound fertilizer, the method including gas chromatography and liquid chromatography, the gas chromatography method for determining the carvacrol content in compound fertilizer includes the following steps:

[0009] S1. Sample pretreatment: Grind the solid compound fertilizer sample through a 0.25mm sieve, weigh 2.00±0.001g, add 15mL of ethyl acetate, vortex for 1min, then ultrasonically extract for 20min, control the temperature ≤40℃, dehydrate with 2g of anhydrous sodium sulfate, centrifuge at 10000r / min for 10min and take the supernatant.

[0010] S2. Matrix purification: The supernatant is passed through a silica gel solid-phase extraction column at a concentration of 500 mg / 6 mL. After activation with 5 mL of ethyl acetate and equilibration with 5 mL of n-hexane, the supernatant is eluted with 10 mL of ethyl acetate, and the eluent is collected.

[0011] S3. Chromatographic analysis: The eluent was diluted to 25 mL with ethyl acetate, filtered through a 0.45 μm organic phase filter membrane, and analyzed using a gas chromatograph equipped with a flame ionization detector (FID). The chromatographic column was an HP-5, 30 m × 0.32 mm × 0.25 μm or a DB-FFAP polar column. The carrier gas was high-purity nitrogen with a purity ≥ 99.999%. The split ratio was 20:1. The column temperature program was: 70 °C for 2 min, then increased to 150 °C at 5 °C / min, and then increased to 250 °C at 20 °C / min for 5 min.

[0012] S4. Quantitative calculation: Establish an external standard curve R based on 0.5-20.0 mg / L carvacrol standard solutions. 2 For samples with a concentration ≥0.999, the content is calculated using the formula X=(C×V×f) / m, where C is the sample solution concentration in mg / L, V is the final volume in mL, f is the dilution factor, and m is the sample mass in g.

[0013] A preferred method for determining the content of carvacrol in compound fertilizer by high performance liquid chromatography is characterized by comprising the following steps:

[0014] A1. Sample pretreatment: Grind the solid compound fertilizer sample through a 0.25mm sieve, weigh 1.00-5.00g, add 15mL of methanol-water mixture, vortex for 1min, then ultrasonically extract for 20min, centrifuge at 10000r / min for 10min and take the supernatant.

[0015] A2. Selective purification: The supernatant was purified by C18 solid phase extraction column with 500 mg / 6 mL of methanol, activated sequentially with 5 mL of methanol and 5 mL of ultrapure water, and then loaded onto the sample. Elution was performed with 10 mL of methanol-water, and the eluent was collected.

[0016] A3. Chromatographic analysis: The eluent was diluted to 25 mL with methanol-water, filtered through a 0.45 μm mixed filter membrane, and analyzed using a high-performance liquid chromatograph equipped with a UV detector. The chromatographic column was a reversed-phase C18 column, 250 mm × 4.6 mm, 5 μm. The mobile phase was methanol-water, the flow rate was 1.0 mL / min, the detection wavelength was 274 nm, and the column temperature was 30 °C.

[0017] A4. Quantitative Calculation: Based on 0.1-10.0 mg / L carvacrol standard solutions, prepared with methanol-water, establish an external standard curve R. 2 For samples with a concentration ≥0.999, the content is calculated using the formula X=(C×V×f) / m, where C is the sample solution concentration in mg / L, V is the final volume in mL, f is the dilution factor, and m is the sample mass in g.

[0018] Preferably, in step S1, ultrasonic extraction is performed under water bath conditions, with the water bath temperature strictly controlled at 35±2℃ and the ultrasonic power at 200W. The extraction container is operated in the dark throughout the process to prevent photolysis of carvacrol. For high-oil organic fertilizer samples, 0.5g of octadecyl bonded silica gel adsorbent is added to the centrifuged supernatant, and the mixture is vortexed for 5min and then centrifuged again to remove lipid interference.

[0019] Preferably, in the purification process of the silica gel solid phase extraction column in step S2, during the activation stage, 5 mL of ethyl acetate and 5 mL of n-hexane are used for rinsing at a flow rate of 2 mL / min, and during the elution stage, 10 mL of ethyl acetate is controlled to pass through the extraction column at a constant flow rate of 1 mL / min to collect the target component.

[0020] Preferably, the column temperature program of the HP-5 column used in step S3 chromatographic analysis is optimized as follows: initially 70℃ for 2 min, then increased to 120℃ at 3℃ / min, and then increased to 240℃ at 8℃ / min for 7 min, shortening the carvacrol retention time to 8.5±0.3 min; the carrier gas flow rate is set to 1.2 mL / min, the hydrogen flow rate to 40 mL / min, the air flow rate to 400 mL / min, the make-up gas flow rate to 30 mL / min, and the FID detector temperature is increased to 280℃ to enhance response sensitivity.

[0021] Preferably, step S4 involves preparing a five-point calibration curve with concentration gradients of 0.1 mg / L, 1.0 mg / L, 5.0 mg / L, 15.0 mg / L, and 30.0 mg / L, and fitting the curve with a quadratic polynomial. 2 ≥0.995.

[0022] Preferably, the volume ratio of the methanol-water mixture in step A1 is dynamically adjusted according to the fertilizer type, with a ratio of 70:30 (v / v) for compound fertilizer and 80:20 (v / v) for organic fertilizer; 0.5g of disodium ethylenediaminetetraacetate is added to high-salt foliar fertilizer to eliminate interference from metal ions.

[0023] Preferably, the activation process of the C18 solid phase extraction column in step A2 is enhanced by sequentially rinsing with 5 mL of methanol, 5 mL of methanol-water (50:50, v / v), and 5 mL of ultrapure water at a flow rate of 1 mL / min, and then using 9 mL of methanol-5% formic acid aqueous solution (80:20, v / v) as the eluent to improve the phenol recovery rate.

[0024] Preferably, step A3 chromatographic analysis uses a diode array detector (DAD) and performs ultraviolet spectral verification simultaneously on the basis of retention time qualitative analysis. It is required that the absorption spectrum matching degree between the sample peak and the standard in the 270-280nm band is ≥98%.

[0025] Preferably, for organic fertilizers containing humic acid, 0.1g of polyethylene glycol-modified magnetic Fe3O4 nanoparticles are added to the extract in step A1. After magnetic separation, the supernatant is taken and directly diluted to volume, eliminating the solid-phase extraction step.

[0026] The technical effects and advantages of this invention are as follows:

[0027] The GC method uses a silica SPE column instead of a traditional diatomaceous earth column and enhances the adsorption of polar impurities through ethyl acetate-n-hexane gradient activation; the HPLC method introduces sodium chloride salting-out and EDTA complexation of metal ions, which reduces impurity peaks in the GC chromatogram, improves the resolution of the carvacrol peak, and extends the HPLC column life.

[0028] The GC method employs temperature-controlled ultrasound at 35±2℃ and complete light-protection operation; the HPLC method uses a room-temperature methanol-water system for extraction, which improves the recovery rate of the GC method; the HPLC method avoids the 60℃ concentration requirement of acetonitrile extraction and improves photostability.

[0029] HPLC optimization of methanol-water method disrupts the hydrogen bond network of fertilizer coating; GC method with the addition of octadecyl-bonded silica gel adsorbent dissociates lipid inclusions, increasing the extraction and recovery rate of coated compound fertilizer; magnetic nanoparticle purification shortens the pretreatment time of organic fertilizer. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention proposes a method for determining the carvacrol content in compound fertilizers. The method includes gas chromatography and liquid chromatography. The gas chromatography method for determining the carvacrol content in compound fertilizers includes the following steps:

[0032] S1. Sample pretreatment: Grind the solid compound fertilizer sample through a 0.25mm sieve, weigh 2.00±0.001g, add 15mL of ethyl acetate, vortex for 1min, and then ultrasonically extract for 20min, controlling the temperature ≤40℃. Dehydrate with 2g of anhydrous sodium sulfate, centrifuge at 10000r / min for 10min, and collect the supernatant. In step S1, ultrasonic extraction is carried out under water bath conditions, with the water bath temperature strictly controlled at 35±2℃ and the ultrasonic power at 200W. The extraction container is operated in the dark throughout the process to prevent photolysis of carvacrol. For high-oil organic fertilizer samples, add 0.5g of octadecyl bonded silica gel adsorbent to the centrifuged supernatant, vortex for 5min, and then centrifuge again to remove lipid interference.

[0033] S2. Matrix purification: The supernatant is passed through a silica gel solid-phase extraction column at a rate of 500 mg / 6 mL, activated sequentially with 5 mL of ethyl acetate and equilibrated with 5 mL of n-hexane, and then eluted with 10 mL of ethyl acetate. The eluent is collected. In the purification process of the silica gel solid-phase extraction column in step S2, during the activation stage, 5 mL of ethyl acetate and 5 mL of n-hexane are used sequentially at a flow rate of 2 mL / min. During the elution stage, 10 mL of ethyl acetate is controlled to pass through the extraction column at a constant flow rate of 1 mL / min to collect the target component.

[0034] S3. Chromatographic analysis: The eluent was diluted to 25 mL with ethyl acetate, filtered through a 0.45 μm organic phase filter membrane, and analyzed using a gas chromatograph equipped with a flame ionization detector (FID). The chromatographic column was an HP-5, 30 m × 0.32 mm × 0.25 μm or a DB-FFAP polar column. The carrier gas was high-purity nitrogen with a purity ≥ 99.999%. The split ratio was 20:1. The column temperature program was: 70 °C for 2 min, then increased to 150 °C at 5 °C / min, and then increased to 250 °C at 20 °C / min for 5 min. The column temperature program for the HP-5 column used in step S3 chromatographic analysis was optimized as follows: initially 70℃ for 2 min, then increased to 120℃ at 3℃ / min, and then increased to 240℃ at 8℃ / min for 7 min, shortening the carvacrol retention time to 8.5±0.3 min; the carrier gas flow rate was set to 1.2 mL / min, the hydrogen flow rate to 40 mL / min, the air flow rate to 400 mL / min, the make-up gas flow rate to 30 mL / min, and the FID detector temperature was increased to 280℃ to enhance response sensitivity.

[0035] S4. Quantitative calculation: Establish an external standard curve R based on 0.5-20.0 mg / L carvacrol standard solutions. 2For samples with a concentration ≥0.999, the content is calculated using the formula X=(C×V×f) / m, where C is the sample solution concentration (mg / L), V is the final volume (mL), f is the dilution factor, and m is the sample mass (g). Step S4 involves preparing a five-point calibration curve with concentration gradients of 0.1 mg / L, 1.0 mg / L, 5.0 mg / L, 15.0 mg / L, and 30.0 mg / L. A quadratic polynomial is used to fit the R-squared value. 2 ≥0.995.

[0036] In addition, the high-performance liquid chromatography method for determining the content of carvacrol in compound fertilizer is characterized by including the following steps:

[0037] A1. Sample pretreatment: Grind the solid compound fertilizer sample through a 0.25mm sieve, weigh 1.00-5.00g, add 15mL of methanol-water mixture, vortex for 1min, then ultrasonically extract for 20min, centrifuge at 10000r / min for 10min and collect the supernatant; The volume ratio of methanol-water mixture in step A1 is dynamically adjusted according to the fertilizer type, using 70:30 (v / v) for compound fertilizer and 80:20 (v / v) for organic fertilizer; Add 0.5g of disodium ethylenediaminetetraacetate to high-salt foliar fertilizer to eliminate metal ion interference.

[0038] A2. Selective purification: The supernatant is purified by a C18 solid-phase extraction column at 500 mg / 6 mL, activated sequentially with 5 mL methanol and 5 mL ultrapure water, and then loaded onto the column. Elution is performed with 10 mL methanol-water, and the eluent is collected. The activation process of the C18 solid-phase extraction column in step A2 is enhanced by sequentially rinsing with 5 mL methanol, 5 mL methanol-water (50:50, v / v), and 5 mL ultrapure water at a flow rate of 1 mL / min. The eluent is changed to 9 mL methanol-5% formic acid aqueous solution (80:20, v / v) to improve the phenol recovery rate.

[0039] A3. Chromatographic Analysis: The eluent was diluted to 25 mL with methanol-water, filtered through a 0.45 μm mixed filter membrane, and analyzed using a high-performance liquid chromatograph equipped with a UV detector. The chromatographic column was a reversed-phase C18 column, 250 mm × 4.6 mm, 5 μm. The mobile phase was methanol-water, the flow rate was 1.0 mL / min, the detection wavelength was 274 nm, and the column temperature was 30 °C. In step A3, the chromatographic analysis was performed using a diode array detector (DAD). In addition to the qualitative analysis based on retention time, UV spectroscopy verification was performed simultaneously. It was required that the absorption spectrum matching degree between the sample peak and the standard in the 270-280 nm band was ≥98%.

[0040] A4. Quantitative Calculation: Based on 0.1-10.0 mg / L carvacrol standard solutions, prepared with methanol-water, establish an external standard curve R. 2For samples with a concentration ≥0.999, the content is calculated using the formula X=(C×V×f) / m, where C is the sample solution concentration in mg / L, V is the final volume in mL, f is the dilution factor, and m is the sample mass in g.

[0041] For organic fertilizers containing humic acid, 0.1g of polyethylene glycol-modified magnetic Fe3O4 nanoparticles are added to the extract in step A1. After magnetic separation, the supernatant is taken and the volume is directly adjusted, eliminating the solid-phase extraction step.

[0042] Before instrumental analysis, a matrix matching standard solution step was added, in which pure carvacrol was diluted with blank compound fertilizer matrix extract to eliminate matrix enhancement / inhibition effects.

[0043] For liquid fertilizer samples, take 5.00 mL of the original solution, add 2 g of anhydrous sodium sulfate directly to dehydrate, purify with silica gel column, and then test according to the standard procedure. In the calculation formula, m is the sampling volume (mL).

[0044] Based on the above, the following embodiments are possible:

[0045] Example 1

[0046] Determination of carvacrol in compound fertilizer by gas chromatography (GC)

[0047] Sample: NPK15-15-15 compound fertilizer (18% organic matter content, 0.5% coated carvacrol)

[0048] Step Details and Results: Pretreatment Optimization: Grind through a 0.25mm sieve, then add 2.00g sample to 15mL ethyl acetate; Temperature-Controlled Ultrasonic Treatment: 35±2℃ water bath, 200W power, operation in the dark; Comparative Experiment: The carvacrol degradation rate in the group treated above 40℃ reached 17%, while this method only reached 3%; Elimination of High Oil Interference: Add 0.5g octadecyl-bonded silica gel, and after vortex centrifugation, the lipid removal rate was 98.5%, and the baseline noise of the GC spectrum was reduced to 50μV (original method >200μV); Silica Gel Column Purification: Gradient elution (ethyl acetate-n-hexane-ethyl acetate), flow rate 1mL / min, humic acid residue <0.1μg / g, recovery rate 95.2±1.8%; Chromatographic Condition Upgrade: Optimized column temperature program, carvacrol retention time 8.3min; FID 280℃ Detection: Signal-to-noise ratio (S / N) improved to 285:1; Quantitative Calibration: 0.1-30mg / L quadratic fitting curve, R 2 =0.9972, with a detection limit (LOD) of 0.02 mg / kg.

[0049] The reagents and materials used in the above methods are shown in Table 1 below:

[0050] Table 1. Reagents and Materials

[0051]

[0052] The GC-FID detection conditions are shown in Table 2 below:

[0053] Table 2 GC-FID Detection Conditions

[0054]

[0055] Example 2

[0056] Determination of carvacrol in organic fertilizers by high performance liquid chromatography (HPLC)

[0057] Sample: Well-rotted chicken manure organic fertilizer (65% organic matter, 28% humic acid content)

[0058] Steps and results:

[0059] The extraction system was dynamically adjusted. With methanol-water (80:20, v / v), the carvacrol dissolution rate was 92.7% (compared to only 78% in the 70:30 system); 0.5g of EDTA-Ca was added. 2+ Complexation rate 99.3%, no clogging in 200 injections of the chromatographic column;

[0060] Magnetic nanoparticle purification: 0.1g PEG-Fe3O4 is added to the extract, and magnetic separation is performed for 2 minutes, eliminating the SPE step and shortening the pretreatment time to 25 minutes. The humic acid removal rate is 99.8%, and there is no interference in the ultraviolet spectrum.

[0061] C18 column co-purification, methanol-5% formic acid elution, phenol recovery rate 96.4% (88% with pure methanol elution);

[0062] DAD spectral verification, with a spectral matching rate of 99.1% in the 270-280nm range, avoids false positives (the false detection rate of traditional UV detection is 15%).

[0063] The matrix effect was eliminated, the blank matrix was matched to the standard curve, and the matrix inhibition effect decreased from -25% to -3.2%.

[0064] The reagents and materials used in the above methods are shown in Table 3 below:

[0065] Table 3. Reagents and Materials

[0066]

[0067] The HPLC detection conditions are shown in Table 4 below.

[0068] Table 4. HPLC Detection Conditions

[0069]

[0070] Based on the two sets of examples above, the characteristics of GC and HPLC methods are compared as shown in Table 5 below.

[0071] Table 5 Comparison of characteristics between GC and HPLC methods

[0072]

[0073] In summary, the GC method in this invention uses a silica SPE column instead of a traditional diatomaceous earth column and enhances the adsorption of polar impurities through ethyl acetate-n-hexane gradient activation; the HPLC method introduces sodium chloride salting-out and EDTA complexation of metal ions, which reduces impurity peaks in the GC chromatogram, improves the resolution of the carvacrol peak, and extends the HPLC column life.

[0074] The GC method employs temperature-controlled ultrasound at 35±2℃ and complete light-protection operation; the HPLC method uses a room-temperature methanol-water system for extraction, which improves the recovery rate of the GC method; the HPLC method avoids the 60℃ concentration requirement of acetonitrile extraction and improves photostability.

[0075] HPLC optimization of methanol-water method disrupts the hydrogen bond network of fertilizer coating; GC method with the addition of octadecyl-bonded silica gel adsorbent dissociates lipid inclusions, increasing the extraction and recovery rate of coated compound fertilizer; magnetic nanoparticle purification shortens the pretreatment time of organic fertilizer.

[0076] Finally, it should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the content of carvacrol in compound fertilizer, characterized in that, The determination methods include gas chromatography and liquid chromatography. The gas chromatography method for determining the content of carvacrol in compound fertilizer includes the following steps: S1. Sample pretreatment: Grind the solid compound fertilizer sample through a 0.25mm sieve, weigh 2.00±0.001g, add 15mL of ethyl acetate, vortex for 1min, then ultrasonically extract for 20min, control the temperature ≤40℃, dehydrate with 2g of anhydrous sodium sulfate, centrifuge at 10000r / min for 10min and take the supernatant. S2. Matrix purification: The supernatant is passed through a silica gel solid-phase extraction column at a concentration of 500 mg / 6 mL. After activation with 5 mL of ethyl acetate and equilibration with 5 mL of n-hexane, the supernatant is eluted with 10 mL of ethyl acetate, and the eluent is collected. S3. Chromatographic analysis: The eluent was diluted to 25 mL with ethyl acetate, filtered through a 0.45 μm organic phase filter membrane, and analyzed using a gas chromatograph equipped with a flame ionization detector (FID). The chromatographic column was an HP-5, 30 m × 0.32 mm × 0.25 μm or a DB-FFAP polar column. The carrier gas was high-purity nitrogen with a purity ≥ 99.999%. The split ratio was 20:

1. The column temperature program was: 70 °C for 2 min, then increased to 150 °C at 5 °C / min, and then increased to 250 °C at 20 °C / min for 5 min. The column temperature program for the HP-5 column used in step S3 chromatographic analysis was optimized as follows: initial temperature of 70℃ for 2 min, then increased to 120℃ at 3℃ / min, and then increased to 240℃ at 8℃ / min for 7 min, shortening the carvacrol retention time to 8.5±0.3 min; the carrier gas flow rate was set to 1.2 mL / min, hydrogen flow rate to 40 mL / min, air flow rate to 400 mL / min, and make-up gas flow rate to 30 mL / min; the FID detector temperature was increased to 280℃ to enhance response sensitivity. S4. Quantitative calculation: Establish an external standard curve R based on 0.5-20.0 mg / L carvacrol standard solutions. 2 For samples with a concentration ≥0.999, the content is calculated using the formula X=(C×V×f) / m, where C is the sample solution concentration in mg / L, V is the final volume in mL, f is the dilution factor, and m is the sample mass in g.

2. The method for determining the carvacrol content in compound fertilizer according to claim 1, characterized in that, A high-performance liquid chromatography method for determining the content of carvacrol in compound fertilizers includes the following steps: A1. Sample pretreatment: Grind the solid compound fertilizer sample through a 0.25mm sieve, weigh 1.00-5.00g, add 15mL of methanol-water mixture, vortex for 1min, then ultrasonically extract for 20min, centrifuge at 10000r / min for 10min and take the supernatant. A2. Selective purification: The supernatant was purified by C18 solid phase extraction column with 500 mg / 6 mL of methanol, activated sequentially with 5 mL of methanol and 5 mL of ultrapure water, and then loaded onto the sample. Elution was performed with 10 mL of methanol-water, and the eluent was collected. A3. Chromatographic analysis: The eluent was diluted to 25 mL with methanol-water, filtered through a 0.45 μm mixed filter membrane, and analyzed using a high-performance liquid chromatograph equipped with a UV detector. The chromatographic column was a reversed-phase C18 column, 250 mm × 4.6 mm, 5 μm. The mobile phase was methanol-water, the flow rate was 1.0 mL / min, the detection wavelength was 274 nm, and the column temperature was 30 °C. A4. Quantitative Calculation: Based on 0.1-10.0 mg / L carvacrol standard solutions, prepared with methanol-water, establish an external standard curve R. 2 For samples with a concentration ≥0.999, the content is calculated using the formula X=(C×V×f) / m, where C is the sample solution concentration in mg / L, V is the final volume in mL, f is the dilution factor, and m is the sample mass in g.

3. The method for determining the carvacrol content in a compound fertilizer according to claim 1, characterized in that, In step S1, ultrasonic extraction was carried out under water bath conditions, with the water bath temperature strictly controlled at 35±2℃ and the ultrasonic power at 200W. The extraction container was operated in the dark throughout the process to prevent photolysis of carvacrol. For high-oil organic fertilizer samples, 0.5g of octadecyl bonded silica gel adsorbent was added to the centrifuged supernatant, and the mixture was vortexed for 5min and then centrifuged again to remove lipid interference.

4. The method for determining the carvacrol content in a compound fertilizer according to claim 1, characterized in that, In the purification process of the silica gel solid phase extraction column in step S2, during the activation stage, 5 mL of ethyl acetate and 5 mL of n-hexane are used for rinsing at a flow rate of 2 mL / min. During the elution stage, 10 mL of ethyl acetate is controlled to pass through the extraction column at a constant flow rate of 1 mL / min to collect the target component.

5. The method for determining the carvacrol content in a compound fertilizer according to claim 1, characterized in that... Step S4 involves preparing a five-point calibration curve with concentration gradients of 0.1 mg / L, 1.0 mg / L, 5.0 mg / L, 15.0 mg / L, and 30.0 mg / L. A quadratic polynomial is used to fit the R-squared value. 2 ≥0.

995.

6. The method for determining the carvacrol content in a compound fertilizer according to claim 2, characterized in that, In step A1, the volume ratio of the methanol-water mixture is dynamically adjusted according to the type of fertilizer. For compound fertilizer, a methanol-water mixture with a volume ratio of 70:30 is used, and for organic fertilizer, a methanol-water mixture with a volume ratio of 80:20 is used. For high-salt foliar fertilizer, 0.5g of disodium ethylenediaminetetraacetate is added to eliminate interference from metal ions.

7. The method for determining the carvacrol content in a compound fertilizer according to claim 2, characterized in that, The activation process of the C18 solid phase extraction column in step A2 is enhanced as follows: 5 mL of methanol, 5 mL of methanol-water solution with a volume ratio of 50:50, and 5 mL of ultrapure water are used sequentially at a flow rate of 1 mL / min. The eluent is then changed to 9 mL of methanol-5% formic acid aqueous solution with a volume ratio of 80:20 to improve the phenol recovery rate.

8. The method for determining the carvacrol content in a compound fertilizer according to claim 2, characterized in that, Step A3 chromatographic analysis uses a diode array detector. Based on retention time qualitative analysis, ultraviolet spectroscopy verification is performed simultaneously, requiring that the absorption spectrum matching degree between the sample peak and the standard in the 270-280nm band be ≥98%.

9. The method for determining the carvacrol content in a compound fertilizer according to claim 2, characterized in that, For organic fertilizers containing humic acid, 0.1g of polyethylene glycol-modified magnetic Fe3O4 nanoparticles are added to the extract in step A1. After magnetic separation, the supernatant is taken and directly diluted to volume, eliminating the solid-phase extraction step.

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