CN109142589B - Method for determining tetramycin residual quantity in soil by using high performance liquid chromatograph - Google Patents
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a method for determining tetramycin residue in soil by using a high performance liquid chromatograph, which comprises the steps of adding a mixed solution of methanol and a phosphate buffer solution into a soil sample, carrying out vibration extraction, then carrying out vacuum filtration by using a Buchner funnel, washing residues twice by using the mixed solution of the methanol and the phosphate buffer solution, collecting all filtrate, transferring the filtrate into a beaker, concentrating the filtrate on a water bath pot until the filtrate is nearly dry, carrying out volume fixing by using the phosphate buffer solution, filtering the filtrate through a microporous filter membrane to obtain a solution to be determined of the soil sample, then determining the solution by using an ultraviolet detector of the high performance liquid chromatograph, and quantifying by using an external standard method. The sample preparation method is simple, the used organic reagents are less, and the sensitivity, accuracy and precision of the method meet the technical requirements of pesticide residue analysis. The invention has the advantages of average recovery rate of 79.74-81.26%, relative standard deviation of 3.68-7.03%, and minimum detection concentration of 0.05mg/kg, and has the advantages of simple operation, low cost, rapidness, high sensitivity, and accurate qualitative and quantitative determination.
Description
Technical Field
The invention relates to detection of tetramycin residual quantity in soil, in particular to a method for determining the tetramycin residual quantity in soil by using a high performance liquid chromatograph.
Background
Tetramycin (Tetramycin) is a novel agricultural antibiotic, is a fermentation metabolite of streptomyces hygroscopicus paraspora subspecies, and comprises four components of A1, A2, B and C, wherein A1 and A2 are macrolide tetraene antibiotics; b is a peptide pyrimidine nucleotide antibiotic; c is a nitrogen heterocyclic aromatic antibiotic. Tetramycin has systemic bacteriostatic activity and the functions of preventing the invasion and expansion of pathogenic bacteria, is a high-efficiency low-toxicity biological pesticide for preventing and treating fungal diseases, has a wide bactericidal spectrum, has extremely strong killing effects on twenty-six known pathogenic fungi such as flagellate fungi, ascomycetes and deuteromycotina fungi, and is suitable for preventing and treating various fungal and bacterial diseases of various crops. At present, the pesticide is mainly applied to preventing and treating diseases of rice and fruit trees in production, and has good prevention and treatment effects. In recent years, with the increasing annual incidence of plant bacterial diseases and the increasing mixed emergence of true bacteria and bacteria, the control advantage of tetramycin is more prominent, the tetramycin has high activity for controlling the bacterial diseases, and simultaneously has control effect on the true bacteria and the bacteria, and the mixing property of the tetramycin and other pesticides is very strong, so that the tetramycin is more and more widely applied.
The soil is used as a complex multi-medium system and is an important destination place of the pesticide. In whatever manner the pesticide is used, it enters directly or indirectly into the soil, which becomes an accumulation reservoir and dissipation ground for the pesticide.
With the application of tetramycin, the residue of tetramycin in soil may threaten the ecological balance, and in order to research the environmental behavior, stability and residue degradation rule of tetramycin in soil, a method for detecting the tetramycin residue in soil must be established. At present, no detection method for the residual quantity of tetramycin in any matrix sample is reported at home and abroad.
Disclosure of Invention
The invention aims to provide a method for determining the residual quantity of tetramycin in soil by using a high performance liquid chromatograph, which has the characteristics of simplicity, accuracy, rapidness, reliability, low cost and easiness in mastering and popularization, and provides a rapid and reliable method for analyzing the residual quantity of tetramycin in soil.
The invention provides a method for determining the residual quantity of tetramycin in soil by using a high performance liquid chromatograph, which comprises the following steps:
the method comprises the following steps: selecting high performance liquid chromatograph detection conditions according to the properties of the tetramycin A1 as follows: the detector adopts an ultraviolet detector; the chromatographic column is Agilent Zorbax-C with length of 250mm × 4.6mm and particle size of 5u18And (3) a chromatographic column, wherein the column temperature is 35 ℃, the flow rate is 0.4mL/min, the sample injection amount is 20uL, and the mobile phase is methanol: 1% by mass aqueous formic acid solution, methanol: the volume ratio of the 1% formic acid aqueous solution is 15: 85, the wavelength is 292nm, and the retention time is 13.5 min;
step two: sample pretreatment
Removing impurities in the collected soil, sieving the impurities by a 20-mesh sieve, uniformly mixing, reserving 200-300g of sample by adopting a four-component sampling method, and putting the sample into a sample container to be used as a soil sample;
step three: extraction of tetramycin A1
Adding methanol (V): shaking and extracting with phosphate buffer solution (V) ═ 3:7 at 35 deg.C for 30min, vacuum filtering with Buchner funnel, and adding methanol (V): washing residues twice by using a mixed solution of 3:7 phosphoric acid buffer solution (V), collecting all filtrate, transferring the filtrate into a beaker, concentrating the filtrate on a water bath kettle at 80 ℃ until the filtrate is nearly dry, metering volume to V mL by using the phosphoric acid buffer solution, and filtering the solution through a 0.45-micrometer microporous filter membrane to obtain a solution to be detected of a soil sample;
step four: calculation of residual quantity
Performing HPLC determination on the soil sample solution to be detected obtained in the step three by using the detection conditions of the high performance liquid chromatograph selected in the step one, recording chromatographic peak retention time and chromatographic peak area, and determining the chromatographic peak area A of tetramycin A1 in the soil sample solution to be detected after the chromatographic peak retention time is determined qualitatively2Substituting the residue into a residue calculation formula to obtain the mass concentration X of the tetramycin A1 in the solution to be detected of the soil sample, and obtaining the residue of the tetramycin A1 in the soil sample, wherein the residue calculation formula is as follows:
in the formula: x-the residual amount of tetramycin A1 in the soil sample, mg/kg;
c-concentration of tetramycin A1 in standard working solution, mg/L;
v, the final constant volume of the sample, mL;
A1-chromatographic peak area for tetramycin a1 in standard working solution;
A2-chromatographic peak area of tetramycin a1 in the sample test solution;
m-sample mass, g.
The method for determining the residual quantity of the tetramycin in the soil by using the high performance liquid chromatograph further comprises the step of determining whether the detection conditions of the high performance liquid chromatograph selected in the step one can carry out qualitative and quantitative analysis on the soil sample before the step two:
dissolving and preparing a standard mother solution with the mass concentration of the tetramycin A1 being 1000mg/L by chromatographic methanol, diluting the standard mother solution by a phosphate buffer salt with the pH value of 7.4 by adopting a gradient dilution method to prepare standard working solutions with the mass concentration of 0.05, 0.1, 0.50, 1.00 and 5.00mg/L, measuring under the detection condition of a selected high performance liquid chromatograph, drawing a standard working curve by taking the mass concentration x of the tetramycin A1 as a horizontal coordinate and the corresponding chromatographic peak area y as a vertical coordinate to obtain a standard working equation of the tetramycin A1 as follows: 37.546x-0.7866, and its correlation coefficient R20.9999, it was determined that the hplc assay conditions selected in step one enabled qualitative and quantitative analysis of the soil samples.
The method for determining the residual quantity of the tetramycin in the soil by using the high performance liquid chromatograph further comprises an adding and recovering experiment after determining that the detection conditions of the high performance liquid chromatograph selected in the step one can carry out qualitative and quantitative analysis on a soil sample, wherein the adding and recovering experiment comprises the following steps:
1) removing impurities in the collected blank soil sample without the tetramycin preparation, sieving the blank soil sample with a 20-mesh sieve, uniformly mixing, and reserving 200g of sample by adopting a quartering sampling method to serve as an addition recovery experiment sample;
2) accurately weighing 10.0g of prepared addition recovery test sample, placing the sample in a 250mL triangular flask with a plug, adding a standard working solution of tetramycin A1 into the addition recovery test sample to ensure that the mass concentration of the tetramycin A1 in the addition recovery test sample is respectively 0.05, 0.50 and 5.00mg/kg, and simultaneously carrying out blank control treatment without containing the tetramycin A1, repeating the addition recovery test sample and the blank sample at each concentration for 5 times, and then adding 40mL of methanol (V): ph7.4 phosphate buffer (V) ═ 3:7, shaking and extracting at 35 ℃ for 30min, followed by suction filtration through a buchner funnel under reduced pressure, followed by 30mL of methanol (V): washing residues with mixed solution of phosphate buffer solution (V) with pH7.4 being 3:7 for two times, collecting all filtrate, transferring into a beaker, concentrating on a water bath kettle at 80 ℃ until the filtrate is nearly dry, fixing the volume to V mL with phosphate buffer solution with pH7.4, and filtering with a 0.45 mu m microporous filter membrane to obtain solution to be detected of the added and recovered experiment sample;
3) carrying out HPLC determination on the solution to be detected of the added and recovered experimental sample obtained in the step 2) by using the detection conditions of the high performance liquid chromatograph selected in the step 1), recording chromatographic peak retention time and chromatographic peak area, and determining the chromatographic peak area A of tetramycin A1 in the solution to be detected of the added and recovered experimental sample after the qualitative determination of the chromatographic peak retention time2Substituting the residual quantity into a residual quantity calculation formula to obtain the mass concentration X of the solution to be detected of the added and recovered experimental sample, namely obtaining the residual quantity of the tetramycin A1 in the added and recovered experimental sample, wherein the residual quantity calculation formula is as follows:
in the formula: x-adding and recovering the residual quantity of tetramycin A1 in the experimental sample, mg/kg;
c-concentration of tetramycin A1 in standard working solution, mg/L;
v, adding a recovered experimental sample to a final constant volume (mL);
A1-chromatographic peak area for tetramycin a1 in standard working solution;
A2addition of test sample to be tested solutionChromatographic peak area for tetramycin a 1;
m-mass of added recovered experimental sample, g;
after the sample preparation in the step 2), the ratio of the residual quantity of the tetramycin A1 in the experimental sample to the quantity of the tetramycin A1 standard substance added is the addition recovery rate, and the addition recovery rate is shown as the following formula:
judging whether the recovery rate reaches 70-110%, whether the relative standard deviation is 0-20%, and whether the minimum detection amount of tetramycin A1 is 1.0 multiplied by 10-9And g, the minimum detection concentration is 0.05mg/kg, whether the correlation coefficient of the linear equation in the step two is greater than 0.99 or not is judged, and if the five indexes all reach the standard, the actual soil sample is detected.
And in the second step, when the soil is collected, randomly selecting 5-10 points, collecting 1-2 kg of soil 0-15 cm below the ground by using a soil drill, and removing broken stones, weeds and plant rhizome impurities in the soil.
The invention has the following beneficial effects:
the invention researches and establishes a sample preparation method for extracting tetramycin in soil by using a mixed solution of methanol and phosphate buffer solution with pH of 7.4 and a detection method by using a high performance liquid chromatography-ultraviolet detector.
Drawings
FIG. 1 is a standard graph of tetramycin A1;
FIG. 2 is a standard solution chromatogram of 1.00mg/L tetramycin A1;
FIG. 3 is a chromatogram of a soil blank sample;
FIG. 4 is a chromatogram of tetramycin A1 with a soil loading concentration of 0.05 mg/kg;
FIG. 5 is a chromatogram of tetramycin A1 with a soil loading concentration of 0.50 mg/kg;
FIG. 6 is a chromatogram of tetramycin A1 with a soil loading concentration of 5.00 mg/kg.
Detailed Description
The following examples are further detailed descriptions of the present invention.
Example 1:
the invention relates to a method for detecting the residual quantity of tetramycin in soil, which comprises the following steps:
1 obtaining of Standard Curve
1.1 liquid chromatography detection conditions
Selecting a 1260 type high performance liquid chromatograph of Agilent company in America according to the property of the tetramycin A1, wherein the detection conditions are as follows: the detector adopts an ultraviolet detector; the chromatographic column is Agilent Zorbax-C with length of 250mm × 4.6mm and particle size of 5u18And (3) a chromatographic column, wherein the column temperature is 35 ℃, the flow rate is 0.4mL/min, the sample injection amount is 20uL, and the mobile phase is methanol: 1% aqueous formic acid, methanol: the volume ratio of the 1% formic acid aqueous solution is 15: 85, the wavelength is 292nm, and the retention time is 13.5 min;
1.2 preparation of Standard solution
Dissolving with chromatographic methanol to prepare a standard mother liquor with the mass concentration of the tetramycin A1 being 1000 mg/L. Standard working solutions of 0.05, 0.1, 0.50, 1.00 and 5.00mg/L were prepared by dilution with phosphate buffer, pH7.4, by the gradient dilution method.
1.3 Standard solution detection and Standard Curve plotting
Standard working solutions of 0.05, 0.1, 0.50, 1.00 and 5.00mg/L were prepared according to the method of 1.2 above, and measured under the selected HPLC detection conditions determined in 1.1 above, and a standard working curve was drawn with the mass concentration (x, mg/L) of tetramycin A1 as abscissa and the corresponding chromatographic peak area (y) as ordinate. The results are shown in FIG. 1, and the standard working equation of tetramycin A1 obtained by statistical analysis is as follows: 37.546x-0.7866 (R)20.9999). The result shows that the mass concentration of the tetramycin A1 and the corresponding chromatographic peak area have good linear relation, so that the method can be used for qualitative and quantitative analysis of samples.
And (5) obtaining the standard curve, and then carrying out a soil sample adding and recycling experiment.
2. Addition recovery experiment
2.1 sample pretreatment
Collecting a blank soil sample without tetramycin preparation, removing impurities such as broken stones, weeds and plant roots and stems in the soil, sieving with a 20-mesh sieve, uniformly mixing, reserving 200g of sample by adopting a quartering method, filling the sample into a plastic sealing bag, and adhering a label.
2.2 sample preparation
Accurately weighing 10.0g of prepared soil sample, placing the soil sample into a 250mL triangular flask with a plug, adding a standard working solution of tetramycin A1 into the soil sample to make the mass concentration of the tetramycin A1 in the soil sample be 0.05, 0.50 and 5.00mg/kg for 3 grades, simultaneously performing blank control treatment on the soil without the tetramycin, repeating the concentration and the blank sample for 5 times, and then adding 40mL of methanol (V) into each sample: ph7.4 phosphate buffer (V) ═ 3:7, shaking and extracting at 35 ℃ for 30min, followed by suction filtration through a buchner funnel under reduced pressure, followed by 30mL of methanol (V): washing the residue twice with a mixed solution of phosphate buffer (pH7.4) (V) ═ 3:7, collecting all the filtrates, transferring into a beaker, concentrating in a water bath (80 ℃) until the filtrates are nearly dry, diluting to 5mL with phosphate buffer (pH7.4), filtering with a 0.45 μm microporous filter membrane to obtain a solution to be tested, and adding the recovered test sample for HPLC detection.
2.3 calculation of residual amount and recovery measurement results
Performing HPLC determination on the solution to be tested of the added recovery test sample obtained in the step 2.2 by using the detection conditions of the high performance liquid chromatograph selected in the step 1.1, recording chromatographic peak retention time and chromatographic peak area, and determining the chromatographic peak area A of the tetramycin A1 in the solution to be tested of the added recovery test sample after the qualitative determination of the chromatographic peak retention time2Substituting the residue into a residue calculation formula to obtain the mass concentration X of the tetramycin A1 in the solution to be tested of the addition recovery test sample, namely obtaining the residue of the tetramycin A1 in the addition recovery test sample, wherein the residue calculation formula is as follows:
in the formula: x-the residual amount of tetramycin A1 in the recovered test sample, mg/kg;
c-concentration of tetramycin A1 in standard working solution, mg/L;
v, adding a recovered experimental sample to a final constant volume of 5 mL;
A1-chromatographic peak area for tetramycin a1 in standard working solution;
A2-adding the chromatographic peak area of tetramycin a1 in the test solution of the recovered test sample;
m-mass of the recovered test sample, g.
After 2.2 samples are prepared, the ratio of the residual quantity of the tetramycin A1 in the test sample to the quantity of the tetramycin A1 standard substance added is the addition recovery rate, and the addition recovery rate is shown as the following formula:
the results of the addition recovery are shown in Table 1.
TABLE 1 recovery and relative standard deviation of tetramycin A1 addition in soil
The residual quantity of the tetramycin A1 in the solution to be tested of the added and recovered test sample is measured by using the HPLC detection conditions selected in the step 1.1, and the average recovery rate is calculated, and the results in the table 1 show that the average addition recovery rate of the tetramycin A1 in the soil is 79.74-81.26%, the relative standard deviation is 3.68-7.03%, and the minimum detection quantity of the tetramycin A1 is 1.0 multiplied by 10-9g, the minimum detection concentration is 0.05mg/kg, the correlation coefficient of the linear equation in the 1.3 is 0.9999, and the sensitivity, the accuracy and the precision of the established detection method are all in accordance with the requirements of pesticide residue detection, and the established method can be usedThe residual amount of tetramycin a1 in soil sprayed or contaminated with tetramycin a1 was tested.
3. Determination of actual samples
(1) Dynamic test for soil digestion of citrus field
In 2016, field test was performed in Huangxingchen, Changsha, Hunan, and a test cell was designed according to the requirement of pesticide residue test criteria, and a 30m test cell was selected2The plots are independently applied, 15% of pyraclostrobin tetramycin suspending agent is applied, the application concentration is 187.5mg/kg of high-dose effective component dosage (the dosage is 800 times of liquid), sampling is carried out 2h, 1, 3, 5, 7, 10, 14, 21, 28, 35, 42 and 60d after application, and clear water blank control is additionally arranged. Collecting a soil sample: randomly taking 5-10 points, collecting 1-2 kg of soil 0-10 cm below the ground by using a soil drill, removing impurities such as broken stones, weeds and plant roots in the soil, uniformly mixing, reserving 300g of sample by adopting a four-point sampling method, and filling the sample into a sample container to be used as a soil sample of the citrus field; then, methanol (V): shaking and extracting with phosphate buffer solution (V) ═ 3:7 at 35 deg.C for 30min, vacuum filtering with Buchner funnel, and adding methanol (V): washing residues twice by using a mixed solution of 3:7 phosphoric acid buffer solution (V), collecting all filtrate, transferring the filtrate into a beaker, concentrating the filtrate on a water bath kettle at 80 ℃ until the filtrate is nearly dry, fixing the volume by using the phosphoric acid buffer solution, and filtering the filtrate through a 0.45-micrometer microporous filter membrane to obtain a solution to be detected of a soil sample; finally, the solution to be detected of the soil sample is detected by using the detection conditions of the high performance liquid chromatograph ultraviolet detector selected in the step 1.1, and the chromatographic peak area A of the tetramycin A1 in the solution to be detected of the soil sample is measured2Substituting into the residue calculation formula, and the result is shown in Table 2.
TABLE 2 digestion dynamic residual amount of tetramycin A1 in soil
(2) Final soil residue test in citrus field
In 2016, a field test was performed in Huangxingchen, Changsha, Hunan, and a test cell was designed according to the requirements of the test criteria for pesticide residue, and a 15% pyraclostrobin tetramycin suspension was prepared at two application doses: low dose and high dose. The low dose is 125mg/kg of the dosage of the effective component of the preparation amount (the preparation amount is 1200 times of the liquid), the high dose is 187.5mg/kg of the dosage of the effective component of the preparation amount (the preparation amount is 800 times of the liquid), the medicine is applied for 3 times, and the interval period of each medicine application is 7 days. Samples of citrus field soil were taken at 14, 21 and 28d after the last application. Each treatment was repeated 3 times with a cell area of 30m2. Additionally setting clear water blank control, and collecting soil samples: randomly taking 5-10 points, collecting 1-2 kg of soil 0-15 cm below the ground by using a soil auger, removing impurities such as broken stones, weeds and plant roots in the soil, uniformly mixing, reserving 300g of sample by using a quartering method, and filling the sample into a sample container to be used as a soil sample of the citrus field; then, methanol (V): shaking and extracting with phosphate buffer solution (V) ═ 3:7 at 35 deg.C for 30min, vacuum filtering with Buchner funnel, and adding methanol (V): washing residues twice by using a mixed solution of 3:7 phosphoric acid buffer solution (V), collecting all filtrate, transferring the filtrate into a beaker, concentrating the filtrate on a water bath kettle at 80 ℃ until the filtrate is nearly dry, fixing the volume by using the phosphoric acid buffer solution, and filtering the filtrate through a 0.45-micrometer microporous filter membrane to obtain a solution to be detected of a soil sample; finally, the solution to be detected of the soil sample is detected by using the detection conditions of the high performance liquid chromatograph ultraviolet detector selected in the step 1.1, and the chromatographic peak area A of the tetramycin A1 in the solution to be detected of the soil sample is measured2Substituting into the residue calculation formula, the results are shown in Table 3.
TABLE 3 Final residual amount of Tetramycin A1 in soil
The above embodiments are provided only for illustrating the technical solutions of the present invention, not for limiting the same, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and to implement the same, and all equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (4)
1. A method for determining the residual quantity of tetramycin in soil by using a high performance liquid chromatograph is characterized by comprising the following steps:
the method comprises the following steps: selecting high performance liquid chromatograph detection conditions according to the properties of the tetramycin A1 as follows: the detector adopts an ultraviolet detector; the chromatographic column is Agilent Zorbax-C with the length of 250mm multiplied by 4.6mm and the particle size of 5um18And (3) a chromatographic column, wherein the column temperature is 35 ℃, the flow rate is 0.4mL/min, the sample injection amount is 20uL, and the mobile phase is methanol: 1% by mass aqueous formic acid solution, methanol: the volume ratio of the 1% formic acid aqueous solution is 15: 85, the wavelength is 292nm, and the retention time is 13.5 min;
dissolving and preparing a standard mother solution with the mass concentration of the tetramycin A1 being 1000mg/L by chromatographic methanol, diluting the standard mother solution by a phosphate buffer salt with the pH value of 7.4 by adopting a gradient dilution method to prepare standard working solutions with the mass concentration of 0.05, 0.1, 0.50, 1.00 and 5.00mg/L, measuring under the detection condition of a selected high performance liquid chromatograph, drawing a standard working curve by taking the mass concentration x of the tetramycin A1 as a horizontal coordinate and the corresponding chromatographic peak area y as a vertical coordinate to obtain a standard working equation of the tetramycin A1 as follows: 37.546x-0.7866, and its correlation coefficient R20.9999, thereby determining that the detection conditions of the high performance liquid chromatograph selected in the step one can carry out qualitative and quantitative analysis on the soil sample;
step two: sample pretreatment
Removing impurities in the collected soil, sieving the impurities by a 20-mesh sieve, uniformly mixing, reserving 200-300g of sample by adopting a four-component sampling method, and putting the sample into a sample container to be used as a soil sample;
step three: extraction of tetramycin A1
Adding a mixed solution of methanol and a phosphate buffer solution in a volume ratio of 3:7 into the soil sample obtained in the step two, oscillating and extracting for 30min at 35 ℃, then performing vacuum filtration through a Buchner funnel, washing residues twice by using the mixed solution of the methanol and the phosphate buffer solution in the volume ratio of 3:7, collecting all filtrate, transferring the filtrate into a beaker, concentrating the filtrate on a water bath kettle at 80 ℃ until the filtrate is nearly dry, fixing the volume of the filtrate to V mL by using the phosphate buffer solution, and filtering the filtrate through a 0.45-micrometer microporous filter membrane to obtain a solution to be detected of the soil sample;
step four: calculation of residual quantity
Performing HPLC determination on the soil sample solution to be detected obtained in the step three by using the detection conditions of the high performance liquid chromatograph selected in the step one, recording chromatographic peak retention time and chromatographic peak area, and determining the chromatographic peak area A of tetramycin A1 in the soil sample solution to be detected after the chromatographic peak retention time is determined qualitatively2Substituting the residue into a residue calculation formula to obtain the mass concentration X of the tetramycin A1 in the solution to be detected of the soil sample, and obtaining the residue of the tetramycin A1 in the soil sample, wherein the residue calculation formula is as follows:
in the formula: x-the residual amount of tetramycin A1 in the soil sample, mg/kg;
c-concentration of tetramycin A1 in standard working solution, mg/L;
v, the final constant volume of the sample, mL;
A1chromatographic peak area of tetramycin a1 in standard working solution;
A2-chromatographic peak area of tetramycin a1 in the sample test solution;
m-sample mass, g.
2. The method of claim 1, further comprising performing an additive recovery test after determining that the conditions of the HPLC test selected in the step of determining whether the qualitative and quantitative analysis of the soil sample can be performed, the additive recovery test comprising the steps of:
1) removing impurities in the collected blank soil sample without the tetramycin preparation, sieving the blank soil sample with a 20-mesh sieve, uniformly mixing, and reserving 200g of sample by adopting a quartering sampling method to serve as an addition recovery experiment sample;
2) accurately weighing 10.0g of prepared addition recovery experiment sample, placing the sample in a 250mL triangular flask with a plug, adding a standard working solution of tetramycin A1 into the addition recovery experiment sample, enabling the mass concentration of tetramycin A1 in the addition recovery experiment sample to be respectively 0.05, 0.50 and 5.00mg/kg, carrying out blank control treatment without tetramycin, repeating the addition recovery experiment sample and the blank sample for 5 times in each concentration, adding 40mL of mixed solution of methanol and phosphate buffer solution with pH7.4 in a volume ratio of 3:7 into each sample, oscillating and extracting for 30min at 35 ℃, then carrying out vacuum filtration by using a Buchner funnel, washing residues twice by using 30mL of mixed solution of methanol and phosphate buffer solution with pH7.4 in a volume ratio of 3:7, collecting all filtrate, transferring the filtrate into a beaker, concentrating the filtrate on a water bath at 80 ℃ until the filtrate is nearly dry, and adding phosphate buffer solution with pH7.4 to V mL, filtering with 0.45 μm microporous membrane to obtain solution to be tested for adding and recovering experimental sample;
3) carrying out HPLC determination on the solution to be detected of the added and recovered experimental sample obtained in the step 2) by using the detection conditions of the high performance liquid chromatograph selected in the step 1), recording chromatographic peak retention time and chromatographic peak area, and determining the chromatographic peak area A of tetramycin A1 in the solution to be detected of the added and recovered experimental sample after the qualitative determination of the chromatographic peak retention time2Substituting the residual quantity into a residual quantity calculation formula to obtain the mass concentration X of the solution to be detected of the added and recovered experimental sample, namely obtaining the residual quantity of the tetramycin A1 in the added and recovered experimental sample, wherein the residual quantity calculation formula is as follows:
in the formula: x-adding and recovering the residual quantity of tetramycin A1 in the experimental sample, mg/kg;
c-concentration of tetramycin A1 in standard working solution, mg/L;
v, adding a recovered experimental sample to a final constant volume (mL);
A1chromatographic peak area of tetramycin a1 in standard working solution;
A2-adding the chromatographic peak area of tetramycin a1 in the test solution of the recovered test sample;
m-mass of added recovered experimental sample, g;
after the sample is prepared in the step 2), the ratio of the residual quantity of the tetramycin A1 in the experimental sample to the quantity of the tetramycin A1 standard substance added is the addition recovery rate, and the addition recovery rate is shown as the following formula:
judging whether the recovery rate reaches 70-110%, whether the relative standard deviation is 0-20%, and whether the minimum detection amount of tetramycin A1 is 1.0 multiplied by 10-9And g, the minimum detection concentration is 0.05mg/kg, whether the correlation coefficient of the linear equation in the step two is greater than 0.99 or not is judged, and if the five indexes all reach the standard, the actual soil sample is detected.
3. The method for measuring the residual amount of tetramycin in soil by using high performance liquid chromatography as claimed in claim 1, wherein the pH value of the phosphate buffer is 7.4.
4. The method for determining the residual quantity of tetramycin in soil by using the high performance liquid chromatograph as claimed in claim 1, wherein 5-10 points are randomly selected during soil collection in the second step, a soil auger is used for collecting 1-2 kg of soil 0-15 cm below the ground, and then broken stones, weeds and plant rhizome impurities in the soil are removed.
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