CN109001334B - Method for measuring residual quantity of benzimidazole drugs in chicken tissues - Google Patents
Method for measuring residual quantity of benzimidazole drugs in chicken tissues Download PDFInfo
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Abstract
The invention discloses a method for measuring the residual quantity of benzimidazole drugs in chicken, chicken liver and chicken kidney. The invention provides a method for simultaneously measuring the residual quantity of nine benzimidazole medicine residual markers in chicken, chicken liver and chicken kidney by adopting a high performance liquid chromatography for the first time, and fills the technical blank in the field. The correlation coefficient of the regression equation of the invention reaches more than 0.999, the determination lower limit is 50 mug/kg, the recovery rate on different addition concentration levels is 75-110%, and the relative standard deviation in a laboratory is less than or equal to 15%. The method solves the technical problem of simultaneously measuring nine benzimidazole medicaments, has originality in the pretreatment technology of samples, particularly in the purification technology, is scientific and accurate in quantification, has strong anti-interference capability and wide substrate application range, and can meet the latest technical requirements of relevant supervision departments in China.
Description
Technical Field
The invention belongs to the technical field of detection of veterinary drug residues in food, and particularly relates to a method for determining residual quantity of benzimidazole drugs in chicken tissues.
Background
The latest national standard for food safety animal food maximum residue limit (draft of comments) in China stipulates benzimidazole drugs including residue markers of febantel, fenbendazole, oxfendazole, oxibendazole, thiabendazole, triclabendazole, albendazole, flubendazole, and mebendazole. The first is that the variety of drugs has increased and the name has been regulated, and the second is that the residue indicator has been greatly modified, in contrast to the previous Ministry of agriculture 235 bulletin. The residual marker of febantel/fenbendazole/oxfendazole is the sum of fenbendazole, oxfendazole and oxfendazole 2-aminosulfone; the residual marker of the oxibendazole is oxibendazole; the residual markers of thiabendazole are thiabendazole and 5-hydroxy thiabendazole; the residual marker of triclabendazole is trichlorophenyl dazole ketone; the residual marker of albendazole is albendazole-2-aminosulfone; the residual marker of the flubendazole is the flubendazole; the residual markers of mebendazole are 2-amino-5-benzoylbenzimidazole and 5-hydroxymebendazole. The modified content is basically consistent with the maximum residual limit requirements of the food code Committee (CAC) and the European Union for benzimidazole drugs.
The issued industry standard and national standard and the published documents only contain one or a plurality of medicines, and a method for completely comprising the nine benzimidazole medicines is not disclosed. The benzimidazole drugs are generally weakly alkaline due to the relationship of chemical structures, but the residual marker trichlorobendazole, namely trichlorobendazole ketone, is a neutral compound, and the simultaneous detection of the residual markers of the nine benzimidazole drugs is required, so that a plurality of technical difficulties exist, and particularly, the high performance liquid chromatography is adopted, so that the requirement on sample purification is higher.
The invention provides a method for simultaneously measuring the residual quantity of nine benzimidazole medicine residual markers in chicken, chicken liver and chicken kidney by adopting a high performance liquid chromatography for the first time, and fills the technical blank in the field. The correlation coefficient of the regression equation of the invention reaches more than 0.999, the determination lower limit is 50 mug/kg, the recovery rate on different addition concentration levels is 75-110%, and the relative standard deviation in a laboratory is less than or equal to 15%. The method solves the technical problem of simultaneously measuring nine benzimidazole medicaments, has originality in the pretreatment technology of samples, particularly in the purification technology, is scientific and accurate in quantification, has strong anti-interference capability and wide substrate application range, and can meet the latest technical requirements of relevant supervision departments in China.
Disclosure of Invention
In view of the technical problem of detection of the existing veterinary drugs, the invention provides a method for rapidly and accurately determining the residual quantity of benzimidazole drugs in chicken tissues.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
A method for measuring the residual quantity of benzimidazole drugs in chicken tissues comprises the following specific steps:
homogenizing, extracting with acetonitrile, purifying the extractive solution with MCX solid phase extraction column, performing rotary evaporation of the purified solution at 38 deg.C under reduced pressure in water bath to 500 μ L, adding 0.1mol/L hydrochloric acid solution to 1mL, vortex for 2min, and centrifuging the solution at 10000r/min for 5 min; then detecting by adopting a high performance liquid chromatograph, and quantifying by adopting an external standard method; the purification method comprises the following steps: taking 5mL of the extracting solution into a 15mL plastic centrifuge tube, adding 3mL of acetonitrile saturated n-hexane, carrying out vortex oscillation for 2min, centrifuging for 5min at 4000r/min, taking the lower layer into a 50mL plastic centrifuge tube, adding 10mL of 0.1mol/L hydrochloric acid solution, uniformly mixing, adding 1mL of n-hexane, carrying out vortex oscillation for 1min, centrifuging for 5min at 10000r/min, discarding the n-hexane at the upper layer, transferring the lower clear solution into a pretreated MCX solid phase extraction column, allowing the sample solution to completely pass at the flow rate of less than 1mL/min, and discarding the effluent liquid; eluting with 3mL of 0.1mol/L hydrochloric acid solution and methanol in a volume ratio of 2:1 and 4mL of 5% ammonia water solution, allowing all eluates to pass through a small column, discarding the eluates, eluting with 4mL of 5% ammonia methanol, allowing all eluates to pass through the small column at a flow rate of 0.5mL/min, and collecting the eluates in a concentration bottle with scales to obtain the final product; the solid phase extraction column comprises: oasis MCX, 60mg, 3 cc; the pretreatment method comprises the following steps: before use, the mixture was activated with 3mL of methanol and 3mL of water in this order.
Further, the extraction method comprises the following steps: weighing 5g of the sample prepared by homogenization, adding 15mL of acetonitrile solution into a 50mL centrifuge tube, carrying out ultrasonic treatment in an ultrasonic water bath for 5min, carrying out vortex oscillation for 15min, centrifuging at 4000r/min for 5min, and transferring the supernatant into a 25mL volumetric flask; and adding 10mL of acetonitrile solution into the residue, placing the mixture in an ultrasonic water bath for ultrasonic treatment for 5min, carrying out vortex oscillation for 10min, centrifuging the mixture for 5min at 4000r/min, combining the supernatant into a 25mL volumetric flask, adding acetonitrile to a constant volume to scale, and shaking the mixture uniformly for later use.
Further, the benzimidazole drugs include: febantel, fenbendazole, oxfendazole, oxibendazole, thiabendazole, triclabendazole, albendazole, flubendazole, and mebendazole; the chicken tissue comprises: chicken, chicken liver and chicken kidney.
Further, the residue markers of the benzimidazole drugs comprise: oxfendazole, fenbendazole, oxfendazole-2-aminosulfone, albendazole-2-aminosulfone, thiabendazole, 5-hydroxythiabendazole, 2-amino-5-benzoylbenzimidazole, 5-hydroxymethylbendazole, flubendazole, oxbendazole, and triclabendazole.
Further, the preparation method of the standard stock solution of the external standard method comprises the following steps: accurately weighing 20mg of oxfendazole, fenbendazole, oxfendazole-2-aminosulfone, albendazole-2-aminosulfone, thiabendazole, 5-hydroxythiabendazole, 2-amino-5-benzoylbenzimidazole, 5-hydroxymebendazole, flubendazole, oxbendazole and trichlorobendazole standard substances which are converted into 100% by mass according to the purity, respectively dissolving the oxfendazole, fenbendazole, 5-hydroxymebendazole, flubendazole, oxbendazole and trichlorobendazole standard substances by using 5mLN and N-dimethylformamide, dissolving the oxfendazole and the trichlorobendazole standard substances by using methanol, and fixing the volume to 200mL to prepare a standard stock solution with the concentration of 200 mg/L; the preparation method of the mixed standard working solution of the external standard method comprises the following steps: respectively measuring 5mL of the standard stock solution in a 100mL volumetric flask, diluting the standard stock solution to a scale with acetonitrile, and preparing a mixed standard working solution with the concentration of 10 mg/L.
Further, the method for preparing the sample comprises the following steps: taking the blank chicken tissue or the edible tissue to be tested, mincing and homogenizing; taking the homogenized test sample as a test sample; taking the homogenized blank sample as a blank sample; taking a blank sample after homogenization, and adding the 10mg/L mixed standard working solution to serve as a blank addition sample; the samples were stored below-18 ℃.
Further, the preparation method of the standard curve of the determination comprises the following steps: precisely measuring a proper amount of the mixed standard working solution, diluting with a methanol-1% acetic acid solution at a ratio of 30:70, v/v, preparing into a series of standard solutions with the concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0mg/L, and measuring by high performance liquid chromatography; drawing a standard curve by taking the measured chromatographic peak area as a vertical coordinate and the corresponding standard solution concentration as a horizontal coordinate, and solving a regression equation and a correlation coefficient; the liquid chromatogram conditions are as follows: a chromatographic column: atlantis T34.6mm × 250mm, particle size 5 μm; mobile phase: a: methanol; b: 1% acetic acid solution, the elution gradient is shown in table 1, and the gradient is linear change; flow rate: 1.0 mL/min; detection wavelength: 292 nm; column temperature: 40 ℃; sample introduction amount: 40 μ L.
Further, the qualitative determination method for determination is as follows: when the drug content in the test sample exceeds the maximum residual limit, the high performance liquid chromatography-diode array detector is used for confirmation; diode array detector spectral conditions: detection start wavelength: 190 nm; end wavelength: 400 nm; slit width: 1.2 nm; under the same test condition, the substance to be detected in the sample has the same retention time with the standard substance detected at the same time, and the deviation is within +/-2.5%; in the spectrogram, the maximum absorption wavelength of the object to be detected and the standard substance is the same, and the deviation of the maximum absorption wavelength and the standard substance is within +/-2 nm; when the relative absorbance of the object to be detected and the standard substance is more than or equal to 10 percent at the wavelength of more than 220nm, the spectrograms of the object to be detected and the standard substance have no obvious difference; the qualitative determination method for determination comprises the following steps: taking the sample solution and the corresponding mixed standard working solution, calibrating a single point or standard curve, and calculating by peak area according to an external standard method; the response values of the benzimidazole medicine residue markers in the mixed standard working solution and the sample solution are within a linear range detected by an instrument; the blank test method comprises the following steps: the parallel operation was carried out using exactly the same procedure except that no sample was added.
Further, the method for calculating and expressing the result of the measurement comprises the following steps: the residual amount of the benzimidazole drug residue marker in the sample is calculated according to formula (1):
in the formula:
x-the residual amount of test substance in the test sample in micrograms per kilogram (μ g/kg);
a- — peak area of the analyte in the sample solution;
cs-concentration of analyte in Standard solutionDegrees in micrograms per liter (μ g/L);
v — volume fixed volume of sample solution in milliliters (mL);
As-peak area of analyte in standard solution;
m-sample mass represented by the sample solution in grams (g).
Further, the sensitivity confirmation method of the measurement method is as follows: determining a quantitative limit based on the value of the signal-to-noise ratio; respectively adding 50 microgram/kg of the benzimidazole medicine residue marker mixed standard working solution into blank chicken, chicken liver and chicken kidney, measuring the ratio of signal to noise, and taking the concentration as the quantitative limit when S/N is more than or equal to 10 and the recovery rate and the relative standard deviation both meet the requirement of the residue detection method; the experimental results are as follows: the limit of quantitation of the method is 50 mug/kg; the accuracy confirmation method of the measuring method comprises the following steps: accurately weighing 5.0g of the blank sample into a 50mL centrifuge tube, adding the series of standard working solutions with known concentrations to prepare tissue samples containing the benzimidazole drug residue markers with the concentrations of 50, 200 and 500 mu g/kg respectively, wherein each concentration is 6 in parallel, and measuring after the sample pretreatment process, and calculating the recovery rate of the added standard; the experimental results are as follows: the recovery rate of the method on the addition concentration level of 50-500 mug/kg is 75-110%; the precision determination method of the measurement method comprises the following steps: accurately weighing 5.0g of the blank sample into a 50mL centrifuge tube, adding the series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of the benzimidazole drug residue markers being 50, 200 and 500 mu g/kg respectively, wherein each concentration is 6 in parallel, processing the tissue samples according to the sample pretreatment process, determining the treated tissue samples, and calculating the indoor relative standard deviation; the experimental results are as follows: the relative standard deviation in the laboratory of the method is less than or equal to 15 percent.
The invention has the following beneficial effects:
the invention provides a method for simultaneously measuring the residual quantity of nine benzimidazole medicine residual markers in chicken, chicken liver and chicken kidney by adopting a high performance liquid chromatography for the first time, and fills the technical blank in the field. Creatively adopts a solid phase extraction columnTwo kinds of compounds with different properties are applied to the column by two different retention mechanisms, so that an ideal purification effect is achieved. For the compound trichlorobenzene dazolone, the N-vinyl pyrrolidone-divinylbenzene copolymer in the solid phase extraction small column packing plays a role of a reverse phase adsorbent; for the remaining ten compounds, the N-vinylpyrrolidone-divinylbenzene copolymer matrix-SO in the column packing was extracted in solid phase3H mainly plays a role of ion exchange. The invention effectively combines the two solutions, and carries out detailed research on the conditions of the pH value of the loading solution, the leaching solution and the elution solution, the percentage of the organic solvent in the solution and the like, thereby obtaining satisfactory results. Although the method is the same as the method for detecting the residual quantity of the benzimidazole medicine in the muscles and livers of the edible animals in the national standard GB/T21324-. The correlation coefficient of the regression equation of the invention reaches more than 0.999, the determination lower limit is 50 mug/kg, the recovery rate on different addition concentration levels is 75-110%, and the relative standard deviation in a laboratory is less than or equal to 15%. The method solves the technical problem of simultaneously measuring eleven residual markers of nine benzimidazole medicaments, has originality in the pretreatment technology of samples, particularly in the extraction and purification technology, is scientific and accurate in quantification, strong in anti-interference capability and wide in matrix application range, and can meet the latest technical requirements of relevant supervision departments in China.
Drawings
FIG. 1 standard solution chromatogram (100. mu.g/L)
FIG. 2 high performance liquid chromatogram of chicken blank sample
FIG. 3 high performance liquid chromatogram of chicken blank addition sample (200. mu.g/L)
FIG. 4 is a high performance liquid chromatogram of chicken liver blank
FIG. 5 is a high performance liquid chromatogram of chicken liver blank addition sample (500. mu.g/L)
FIG. 6 high performance liquid chromatogram of chicken kidney blank sample
FIG. 7 is a high performance liquid chromatogram of chicken kidney blank addition sample (100. mu.g/L)
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the present invention will be described in detail with reference to specific embodiments and drawings, wherein the drawings only apply to the following embodiments, and other drawings can be obtained by a person skilled in the art according to the method of the present invention. The scope of the invention is not limited to the following examples.
Example 1: establishing method
1. Reagents and materials
The reagents used below, unless otherwise noted, are analytical reagents; the water is first-grade water meeting the GB/T6682 specification.
And (3) standard substance: oxfendazole (CAS number: 53716-50-0), Fenbendazole (Fenbendazole, CAS number: 43210-67-9), Oxfendazole-2-aminosulfone (Oxfendazole sulpholane, CAS number: 54029-20-8), Albendazole-2-aminosulfone (Albendazole-2-aminosulfone, CAS number: 80983-34-2), Thiabendazole (Thiabendazole, CAS number: 148-79-8), 5-hydroxythiabendazole (5-hydroxythiabendazole, CAS number: 948-71-0), 2-amino-5-benzoylbenzimidazole (Mebendazole-amine, CAS number: 52329-60-9), 5-hydroxymethylbenzobendazole (5-hydroxyebendazole, CAS number: 60254-357-3595), Fluorobenzobendazole (Fluorobenzobendazole, CAS number: 3115-430-6-15, CAS number: 3195-0), Oxibendazole (CAS number: 20559-55-1) and trichlorobendazole (Ketotriazabendazole, CAS number: 1201920-88-8) with contents of more than 97%.
Acetonitrile: carrying out chromatographic purification; methanol: carrying out chromatographic purification; acetic acid: carrying out chromatographic purification; hydrochloric acid; 25% ammonia water;
0.1mol/L hydrochloric acid solution: measuring 8.3mL of concentrated hydrochloric acid, and adding water to dilute the concentrated hydrochloric acid to 1000 mL;
0.1mol/L hydrochloric acid solution + methanol (2+1, v/v): 200mL of 0.1mol/L hydrochloric acid solution and 100mL of methanol are respectively weighed and mixed uniformly.
5% aqueous ammonia solution: 5mL of 25% ammonia water was measured, diluted with water to 100mL, and prepared immediately before use.
5% ammonia methanol solution: 5mL of 25% ammonia water was measured, diluted to 100mL with methanol, and prepared immediately before use.
1% acetic acid solution: 10mL of acetic acid was measured, diluted with water to 1000mL and prepared immediately before use.
Solid phase extraction column: oasis MCX, 60mg, 3 mL. Before use, the mixture was activated with 3mL of methanol and 3mL of water in this order.
Preparation of standard stock solution: accurately weighing 20mg of oxfendazole, fenbendazole, oxfendazole-2-aminosulfone, albendazole-2-aminosulfone, thiabendazole, 5-hydroxythiabendazole, 2-amino-5-benzoylbenzimidazole, 5-hydroxymebendazole, flubendazole, oxbendazole and trichlorobendazole standard substances which are converted into 100 mass percent according to the purity, respectively dissolving the oxfendazole, fenbendazole, 5-hydroxymebendazole, flubendazole, oxbendazole and trichlorobendazole standard substances in 5mLN and N-dimethylformamide, dissolving the solutions in methanol and fixing the volume to 200mL to prepare a standard stock solution with the concentration of 200 mg/L.
Preparing a mixed standard working solution: respectively measuring 5mL of standard stock solution into a 100mL volumetric flask, diluting the stock solution to a scale with acetonitrile, and preparing into a mixed standard working solution with the concentration of 10 mg/L.
2. Apparatus and instrument
High performance liquid chromatograph: LC-20AD, with autosampler, column oven, UV detector and diode array detector, Shimadzu.
Analytical balance: 0.00001 g of sensitive amount, Sartorius company.
Balance: the amount of the drug was 0.01g, Sartorius Co.
Meat tissue masher: GM 200, Retsch Corp.
A vortex oscillator: MS 3basic, IKA, Inc.
Ultrasonic water bath: SW 30H, SONO SWISS Corp.
A centrifuge: 3-30K, SIGMA corporation.
Reduced pressure rotary evaporator: IKA (R) Inc.
3. Preparation and preservation of samples
3.1 preparation of samples
Taking chicken, chicken liver and chicken kidney blank or edible tissue to be tested, mincing and homogenizing.
Taking the homogenized test sample as a test sample.
Taking a blank sample after homogenizing as a blank sample.
Taking a blank sample after homogenization, and adding a standard working solution with a proper concentration to serve as a blank addition sample.
3.2 preservation of the samples
Storing at below-18 deg.C.
4 measurement step
4.1 extraction
Weighing 5g of sample (accurate to 0.01g), adding 15mL of acetonitrile solution into a 50mL centrifuge tube, carrying out ultrasonic treatment in an ultrasonic water bath for 5min, carrying out vortex oscillation for 15min, centrifuging at 4000r/min for 5min, and transferring the supernatant into a 25mL volumetric flask; and adding 10mL of acetonitrile solution into the residue, placing the mixture in an ultrasonic water bath for ultrasonic treatment for 5min, carrying out vortex oscillation for 10min, centrifuging the mixture for 5min at 4000r/min, combining the supernatant into a 25mL volumetric flask, adding acetonitrile to a constant volume to scale, and shaking the mixture uniformly for later use.
4.2 purification
Taking 5mL of the extracting solution in a 15mL plastic centrifuge tube, adding 3mL of acetonitrile saturated normal hexane, carrying out vortex oscillation for 2min, centrifuging for 5min at 4000r/min, taking the lower layer in a 50mL plastic centrifuge tube, adding 10mL of 0.1mol/L hydrochloric acid solution, uniformly mixing, adding 1mL of normal hexane, carrying out vortex oscillation for 1min, centrifuging for 5min at 10000r/min, discarding the normal hexane at the upper layer, transferring the lower clear solution into a pretreated MCX solid phase extraction column, allowing the sample solution to completely pass at the flow rate of less than 1mL/min, and discarding the effluent liquid. And (3) sequentially eluting with 3mL of 0.1mol/L hydrochloric acid solution, methanol (2+1, v/v) and 4mL of 5% ammonia water solution, allowing all the eluates to pass through a small column, and discarding the eluates. And finally, eluting with 4mL of 5% ammonia methanol, enabling the eluent to completely pass through a small column at the flow rate of 0.5mL/min, collecting the eluent in a concentration bottle with scales, carrying out reduced pressure rotary evaporation at 38 ℃ in a water bath to about 500 mu L, adding 0.1mol/L hydrochloric acid solution to fix the volume to 1mL, carrying out vortex for 2min, and centrifuging the solution at 10000r/min for 5min for determination.
4.3 preparation of Standard Curve
A proper amount of mixed standard working solution is precisely measured, diluted by methanol-1% acetic acid solution (30+70, v/v), and prepared into a series of standard solutions with the concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0mg/L for high performance liquid chromatography determination. And drawing a standard curve by taking the measured chromatographic peak area as a vertical coordinate and the corresponding standard solution concentration as a horizontal coordinate, and solving a regression equation and a correlation coefficient.
4.4 determination
4.4.1 liquid chromatography conditions
A chromatographic column: atlantis T3(4.6 mm. times.250 mm, particle size 5 μm).
Mobile phase: a: methanol; b: 1% acetic acid solution, the elution gradient is shown in Table 1, and the gradient is linear.
TABLE 1 gradient elution procedure
Flow rate: 1.0 mL/min; detection wavelength: 292 nm; column temperature: 40 ℃; sample introduction amount: 40 μ L.
4.4.2 assay
4.4.2.1 qualitative determination
When the drug content in the test sample exceeds the maximum residual limit, the test sample is verified by a high performance liquid chromatography-diode array detector.
Diode array detector spectral conditions: detection start wavelength: 190 nm; end wavelength: 400 nm; slit width: 1.2 nm.
Under the same test conditions, the substance to be detected in the sample has the same retention time as the standard substance detected at the same time, and the deviation is within +/-2.5%. In the spectrogram, the analyte and the standard have the same maximum absorption wavelength, and the deviation of the two is within +/-2 nm. When the relative absorbance of the object to be detected and the standard substance is more than or equal to 10 percent at the wavelength of more than 220nm, the spectrograms of the object to be detected and the standard substance have no obvious difference.
4.4.2.2 quantitative determination
Taking the sample solution and the corresponding standard solution, calibrating a single point or a standard curve, and calculating by peak area according to an external standard method. The response values of the benzimidazole drug residue markers in the standard solution and the sample solution are within the linear range detected by the instrument. Under the chromatographic conditions, the high performance liquid chromatograms of the standard solution, the blank sample and the added sample are shown in the attached drawings.
4.5 blank test
The parallel operation was carried out using exactly the same procedure except that no sample was added.
4.6 results calculation and presentation
The residual amount of the benzimidazole residue marker in the sample is calculated according to the formula (1):
in the formula:
x-the residual amount of test substance in the test sample in micrograms per kilogram (μ g/kg);
a- — peak area of the analyte in the sample solution;
cs-concentration of test substance in standard solution in micrograms per liter (μ g/L);
v — volume fixed volume of sample solution in milliliters (mL);
As-peak area of analyte in standard solution;
m-sample mass represented by the sample solution in grams (g).
4.7 method sensitivity, accuracy and precision
4.7.1 sensitivity
The quantitative limit is determined based on the value of the signal-to-noise ratio (S/N). Respectively adding 50 mu g/kg of benzimidazole drug residue marker standard solution into blank chicken, chicken liver and chicken kidney, measuring the ratio of signal to noise, and determining the concentration as the quantitative limit when S/N is more than or equal to 10 and the recovery rate and the relative standard deviation both meet the requirement of the residue detection method.
The experimental results are as follows: the limit of quantitation of this method is 50. mu.g/kg.
4.7.2 accuracy
Accurately weighing 5.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of benzimidazole drug residue markers of 50, 200 and 500 mug/kg respectively, performing 6 parallels on each concentration, processing according to the sample pretreatment process, determining, and calculating the standard addition recovery rate.
The experimental results are as follows: the recovery rate of the method on the addition concentration level of 50-500 mu g/kg is 75-110%.
4.7.3 precision
Accurately weighing 5.0g of blank sample into a 50mL centrifuge tube, adding a series of standard working solutions with known concentrations to prepare tissue samples with the concentrations of benzimidazole drug residue markers of 50, 200 and 500 mug/kg respectively, performing 6 parallels on each concentration, processing according to the sample pretreatment process, determining, and calculating the indoor relative standard deviation.
The experimental results are as follows: the relative standard deviation in the laboratory of the method is less than or equal to 15 percent.
5 examples of
The method is used for daily detection, and 150 detected chicken, chicken liver and chicken kidney samples are obtained, wherein albendazole-2-aminosulfone is detected in 1 chicken liver, the content of albendazole-2-aminosulfone is 78 mug/kg, and the others are not detected.
Example 2: selection of extraction solvent
The invention compares acetonitrile with alkaline ethyl acetate adopted in GB/T21324-2007 method for detecting the residual quantity of benzimidazole drugs in muscles and livers of edible animals, under the condition of a chromatographic system of the method, the interference peak of an acetonitrile extracting solution is far less than that of the alkaline ethyl acetate, in addition, the alkaline ethyl acetate extracting solution needs to be converted into a solvent before the next step of purification and column loading, nitrogen-blowing concentration or reduced-pressure rotary evaporation concentration is carried out until the solution is dry, and then the acetonitrile-0.1 mol/L hydrochloric acid solution is used for dissolution, the degradation of the trichloro-dazolone can be caused in the concentration process, while the acetonitrile extracting solution does not need to be concentrated, and the column loading can be carried out after the 0.1mol/L hydrochloric acid solution is added. The extraction efficiency of the acetonitrile extracting solution to all compounds reaches more than 95 percent, so the invention selects acetonitrile as an extraction solvent.
Example 3: selection of purification conditions
Firstly, the invention adopts a reversed phase adsorbent HLB solid phase extraction column, when the proportion of acetonitrile in the solution on the column is 10%, oxfendazole-2-amino sulfone and 5-hydroxythiabendazole are lost, so that the acetonitrile extracting solution must be concentrated, thereby not only causing the degradation of the compound to be detected, but also causing the reduction of the recovery rate due to the low proportion of organic solvent during redissolution and poor dissolving effect.
By comparison, the method adopts an MCX solid phase extraction column, and for trichloro-phenyl dazolone, the N-vinyl pyrrolidone-divinylbenzene copolymer in the filler plays a role of a reverse phase adsorbent; for the remaining ten compounds, the N-vinylpyrrolidone-divinylbenzene copolymer matrix-SO 3H in the filler predominantly functions as an ion exchange. The two solutions are combined together, the loading solution, the leaching solution and the elution solution have more condition limitations, and both the pH value of the solution and the percentage of the organic solvent in the solution need to be considered. Treating the labeled sample according to the column passing conditions used in GB/T21324-2007 method for detecting the residual quantity of the benzimidazole drugs in the muscles and livers of the edible animals, wherein the trichloro-phenyl dazolone can be eluted in the step of eluting with methanol, and the final eluent hardly contains the trichloro-phenyl dazolone. The ratio of acetonitrile to 0.1mol/L hydrochloric acid solution in the upper column solution of the method is 1:2, if the ratio is 1:1, the trichloro-phenyl-dazolone can be largely lost and is not retained by a small column; the ratio of 0.1mol/L hydrochloric acid solution in leaching solution to methanol in methanol is not more than 50%, the other leaching solution 5% ammonia water solution can remove impurities between the two peaks of the oxfendazole-2-aminosulfone and the 5-hydroxythiabendazole, and the impurities can not be removed by adopting 0.1mol/L hydrochloric acid solution; the eluent is more suitable for 5% ammonia methanol than 10% ammonia acetonitrile adopted by GB/T21324-2007, if the eluent is eluted by the ammonia acetonitrile, the proportion of ammonia has great influence on the elution capability of the 2-amino-5-benzoyl benzimidazole, the 5% ammonia acetonitrile can only be eluted by about 50%, the 10% ammonia acetonitrile is volatile, the reduction of ammonia can directly influence the recovery rate of the 2-amino-5-benzoyl benzimidazole, the reproducibility of the method is poor, the selected ammonia methanol is more stable, and the ammonia methanol is easier to concentrate.
Claims (8)
1. A method for measuring the residual quantity of benzimidazole drugs in chicken tissues is characterized by comprising the following specific steps: homogenizing, extracting with acetonitrile, purifying the extractive solution, subjecting the purified solution to rotary evaporation under reduced pressure in 38 deg.C water bath to 500 μ L, adding 0.1mol/L hydrochloric acid solution to constant volume of 1mL, vortex for 2min, centrifuging the solution at 10000r/min for 5min, detecting with high performance liquid chromatograph, and quantifying by external standard method; the purification method comprises the following steps: taking 5mL of extracting solution into a 15mL plastic centrifuge tube, adding 3mL of acetonitrile saturated normal hexane, carrying out vortex oscillation for 2min, centrifuging for 5min at 4000r/min, taking the lower layer into a 50mL plastic centrifuge tube, adding 10mL of 0.1mol/L hydrochloric acid solution, uniformly mixing, adding 1mL of normal hexane, carrying out vortex oscillation for 1min, centrifuging for 5min at 10000r/min, discarding the normal hexane at the upper layer, transferring the lower clear solution into a pretreated MCX solid phase extraction column, allowing the sample solution to completely pass at the flow rate of less than 1mL/min, and discarding the effluent; eluting with 3mL of 0.1mol/L hydrochloric acid solution and methanol in a volume ratio of 2:1 and 4mL of 5% ammonia water solution, allowing all eluates to pass through a small column, discarding the eluates, eluting with 4mL of 5% ammonia methanol, allowing all eluates to pass through the small column at a flow rate of 0.5mL/min, and collecting the eluates in a concentration bottle with scales to obtain the final product; the solid phase extraction column comprises: oasis MCX, 60mg, 3 cc; the pretreatment method comprises the following steps: before use, sequentially activating by using 3mL of methanol and 3mL of water; the benzimidazole medicine comprises: febantel, fenbendazole, oxfendazole, oxibendazole, thiabendazole, triclabendazole, albendazole, flubendazole, and mebendazole; the chicken tissue comprises: chicken, chicken liver and chicken kidney; the residue markers of the benzimidazole drugs comprise: oxfendazole, fenbendazole, oxfendazole-2-aminosulfone, albendazole-2-aminosulfone, thiabendazole, 5-hydroxythiabendazole, 2-amino-5-benzoylbenzimidazole, 5-hydroxymethylbendazole, flubendazole, oxbendazole, and triclabendazole.
2. The assay of claim 1, wherein the extraction method is: weighing 5g of the sample prepared by homogenization, adding 15mL of acetonitrile solution into a 50mL centrifuge tube, carrying out ultrasonic treatment in an ultrasonic water bath for 5min, carrying out vortex oscillation for 15min, centrifuging at 4000r/min for 5min, and transferring the supernatant into a 25mL volumetric flask; and adding 10mL of acetonitrile solution into the residue, placing the mixture in an ultrasonic water bath for ultrasonic treatment for 5min, carrying out vortex oscillation for 10min, centrifuging the mixture for 5min at 4000r/min, combining the supernatant into a 25mL volumetric flask, adding acetonitrile to a constant volume to scale, and shaking the mixture uniformly for later use.
3. The assay of claim 1, wherein the standard stock solution of the external standard method is prepared by: accurately weighing 20mg of oxfendazole, fenbendazole, oxfendazole-2-aminosulfone, albendazole-2-aminosulfone, thiabendazole, 5-hydroxythiabendazole, 2-amino-5-benzoylbenzimidazole, 5-hydroxymebendazole, flubendazole, oxbendazole and trichlorobendazole standard substances which are converted into 100% by mass according to the purity, respectively dissolving the oxfendazole, fenbendazole, 5-hydroxymebendazole, flubendazole, oxbendazole and trichlorobendazole standard substances by using 5mLN and N-dimethylformamide, dissolving the oxfendazole and the trichlorobendazole standard substances by using methanol, and fixing the volume to 200mL to prepare a standard stock solution with the concentration of 200 mg/L; the preparation method of the mixed standard working solution of the external standard method comprises the following steps: respectively measuring 5mL of the standard stock solution in a 100mL volumetric flask, diluting the standard stock solution to a scale with acetonitrile, and preparing a mixed standard working solution with the concentration of 10 mg/L.
4. The assay of claim 3, wherein the sample is prepared by: taking chicken tissue blank or edible tissue to be tested, mincing, and homogenizing; taking the homogenized test sample as a test sample; taking the homogenized blank sample as a blank sample; taking a blank sample after homogenization, and adding the 10mg/L mixed standard working solution to serve as a blank addition sample; the samples were stored below-18 ℃.
5. An assay method according to claim 3, wherein the standard curve is prepared by: precisely measuring a proper amount of the mixed standard working solution, diluting with a methanol-1% acetic acid solution at a ratio of 30:70, v/v, preparing into a series of standard solutions with the concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0mg/L, and measuring by high performance liquid chromatography; using the measured chromatographic peak area as ordinate, correspondingThe concentration of the standard solution is an abscissa, a standard curve is drawn, and a regression equation and a correlation coefficient are solved; the liquid chromatography conditions were: a chromatographic column: atlantis T34.6mm × 250mm, particle size 5 μm; mobile phase: a: methanol; b: 1% acetic acid solution, the elution gradient is shown in table 1, and the gradient is linear change;
TABLE 1 gradient elution procedure
Flow rate: 1.0 mL/min; detection wavelength: 292 nm; column temperature: 40 ℃; sample introduction amount: 40 μ L.
6. The assay of claim 5, wherein the qualitative assay of the assay is: when the drug content in the test sample exceeds the maximum residual limit, the high performance liquid chromatography-diode array detector is used for confirmation; diode array detector spectral conditions: detection start wavelength: 190 nm; end wavelength: 400 nm; slit width: 1.2 nm; under the same test condition, the substance to be detected in the sample has the same retention time with the standard substance detected at the same time, and the deviation is within +/-2.5%; in the spectrogram, the maximum absorption wavelength of the object to be detected and the standard substance is the same, and the deviation of the maximum absorption wavelength and the standard substance is within +/-2 nm; when the relative absorbance of the object to be detected and the standard substance is more than or equal to 10 percent at the wavelength of more than 220nm, the spectrograms of the object to be detected and the standard substance have no obvious difference; the quantitative determination method comprises the following steps: taking a sample solution and the corresponding mixed standard working solution, calibrating a single point or standard curve, and calculating by peak area according to an external standard method; the response values of the benzimidazole medicine residue markers in the mixed standard working solution and the sample solution are within a linear range detected by an instrument; the blank test method comprises the following steps: the parallel operation was carried out using exactly the same procedure except that no sample was added.
7. The assay of claim 6, wherein the results of the assay are calculated and expressed by: the residual amount of the benzimidazole drug residue marker in the sample is calculated according to formula (1):
in the formula:
x-the residual amount of test substance in the test sample in micrograms per kilogram (μ g/kg);
a- — peak area of the analyte in the sample solution;
cs-concentration of test substance in standard solution in micrograms per liter (μ g/L);
v — volume fixed volume of sample solution in milliliters (mL);
As-peak area of analyte in standard solution;
m-sample mass represented by the sample solution in grams (g).
8. The method of claim 7, wherein the method of determining the sensitivity comprises: determining a quantitative limit based on the value of the signal-to-noise ratio; respectively adding 50 microgram/kg of benzimidazole medicine residue marker mixed standard working solution into blank chicken, chicken liver and chicken kidney, measuring the ratio of signal to noise, and taking the concentration as the quantitative limit when S/N is more than or equal to 10 and the recovery rate and the relative standard deviation both meet the requirement of the residue detection method; the experimental results are as follows: the limit of quantitation of the method is 50 mug/kg; the accuracy confirmation method of the measuring method comprises the following steps: accurately weighing 5.0g of the blank sample into a 50mL centrifuge tube, adding the series of standard solutions with known concentrations to prepare tissue samples with the concentrations of the benzimidazole drug residue markers being 50, 200 and 500 mu g/kg respectively, wherein each concentration is 6 in parallel, determining after the sample pretreatment process, and calculating the recovery rate of the added standard; the experimental results are as follows: the recovery rate of the method on the addition concentration level of 50-500 mug/kg is 75-110%; the precision determination method of the measurement method comprises the following steps: accurately weighing 5.0g of the blank sample into a 50mL centrifuge tube, adding the series of standard solutions with known concentrations to prepare tissue samples with the concentrations of the benzimidazole drug residue markers being 50, 200 and 500 mu g/kg respectively, wherein each concentration is 6 in parallel, processing and determining the tissue samples according to the sample pretreatment process, and calculating the indoor relative standard deviation; the experimental results are as follows: the relative standard deviation in the laboratory of the method is less than or equal to 15 percent.
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