CN111735883B - Method for detecting phenylbutazone in horse meat based on eutectic solvent - Google Patents
Method for detecting phenylbutazone in horse meat based on eutectic solvent Download PDFInfo
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Abstract
The invention provides a method for detecting phenylbutazone in horse meat based on a eutectic solvent, and belongs to the technical field of component detection. According to the method, an ammonium acetate buffer solution containing ascorbic acid is used as an extraction solvent, the acidic ammonium acetate buffer solution can increase the solubility of the phenylbutazone and improve the extraction efficiency of liquid-liquid microextraction, and the ascorbic acid contained in an extracting solution can avoid the decomposition phenomenon of the phenylbutazone in an acidic solution; meanwhile, the invention uses the ammonium acetate buffer solution containing ascorbic acid as the extraction solvent, thus avoiding the use of a large amount of organic extraction solvent. The invention uses the eutectic solvent as the extractant of liquid-liquid microextraction, can be mutually dissolved with the extraction solvent and quickly reach extraction balance, thereby improving the extraction efficiency. The method can realize the layering of the extracting agent and the extracting solvent by using the polar organic dispersing agent, and has simple operation and short time consumption. The results of the examples show that the detection method provided by the invention has good repeatability and precision.
Description
Technical Field
The invention relates to the technical field of component detection, in particular to a method for detecting phenylbutazone in horse meat based on a eutectic solvent.
Background
Phenylbutazone (phenybutazone), also known as Phenylbutazone, belongs to a non-steroidal anti-inflammatory drug, is invented in the middle of the 20 th century, is mainly used for treating human rheumatoid arthritis and ventilation, and is also used for pain relief and fever reduction of horses. Its side effects are similar to other non-steroidal anti-inflammatory drugs, and excessive or long-term use can cause gastrointestinal ulcers, hematopathy, kidney damage, oral lesions, internal hemorrhage, etc. Furthermore, phenylbutazone has embryotoxicity, and in addition, it may inhibit the regeneration of leukocytes, causing aplastic anemia in humans.
Because of its side effects, the use of the drug by humans has been prohibited internationally and has strict restrictions on its entry into the human food chain, and countries such as the european union, the united states, canada and japan prohibit the use of the drug for food animals. Although strict regulations are imposed in each country, it still happens that horse meat containing phenylbutazone enters the market. In view of the above, it is necessary to establish a fast and simple method for detecting phenylbutazone in horse meat to detect the quality of imported meat.
Aiming at the detection of phenylbutazone in animal muscle tissues, Pongyuan aromatic and the like establish an LC-UV (liquid chromatography-ultraviolet) determination method for the residual amount of phenylbutazone in the muscle tissues of slaughtered animals, and establish GB/T20754-2006 'liquid chromatography-ultraviolet detection method for the residual amount of phenylbutazone in livestock and poultry meat', the detection method uses a large amount of ethyl acetate organic mixed solution as an extraction solvent, uses a solid-phase extraction column for purification, and has more steps and higher cost. SN/T2190-2008 adopts a conventional liquid phase extraction method to detect the non-steroidal anti-inflammatory drugs in the animal-derived matrix, uses a large amount of organic solvents of acetonitrile and n-hexane, and needs the steps of liquid separation, purification, nitrogen blowing concentration and the like during extraction, so that the operation is complicated.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting phenylbutazone in horse meat based on a eutectic solvent. The detection method provided by the invention is simple to operate, the use amount of the organic solvent is small, and the content of the phenylbutazone in the horse meat can be quickly and accurately detected.
In order to achieve the purpose of the invention, the invention provides the following technical scheme:
the invention provides a method for detecting phenylbutazone in horse meat based on a eutectic solvent, which comprises the following steps:
(1) mixing a horse meat sample with an extraction solvent, a water-soluble inorganic salt, a eutectic solvent and a polar organic dispersant, sequentially carrying out liquid-liquid microextraction and centrifugation, taking supernatant liquor to a constant volume, and obtaining a liquid to be detected after the supernatant liquor passes through a membrane;
the extraction solvent is an ammonium acetate buffer solution containing ascorbic acid; the pH value of the ammonium acetate buffer solution containing ascorbic acid is 3-5;
the hydrogen bond acceptor of the eutectic solvent is choline chloride, and the hydrogen bond donor is phenol;
(2) detecting the liquid to be detected by using a high performance liquid chromatography-tandem mass spectrometry method to obtain the peak area of the liquid to be detected;
(3) obtaining the content of phenylbutazone in horse meat according to the standard curve and the peak area obtained in the step (2); the standard curve is a linear relation curve of the concentration of the phenylbutazone standard solution and the peak area of the high performance liquid chromatography-tandem mass spectrum.
Preferably, the molar concentration of the ascorbic acid in the ascorbic acid-containing ammonium acetate buffer solution is 0.005-0.05 mol/L, and the molar concentration of the ammonium acetate is 20 mmol/L.
Preferably, the molar ratio of choline chloride to phenol in the eutectic solvent is 1: 1-4.
Preferably, the water-soluble inorganic salt is sodium chloride, and the polar organic dispersant is one of acetonitrile, acetone and tetrahydrofuran.
Preferably, the horse meat sample, the extraction solvent, the water-soluble inorganic salt, the eutectic solvent and the polar organic dispersant are used in a ratio of 2.5 g: 5mL of: 1.0 g: 0.8 mL: 0.8 mL.
Preferably, the time of the liquid-liquid micro-extraction is 5 min; the rotation speed of the centrifugation is 10000r/min, and the time is 5 min.
Preferably, the detection conditions of the high performance liquid chromatography comprise:
the chromatographic column is Acquity UPLC BEH C18, the length of the chromatographic column is 50mm, the diameter of the chromatographic column is 2.1mm, and the particle size of the filler is 1.7 μm;
the sample injection amount is 5 mu L;
the mobile phase A is an aqueous solution containing 5mmol/L of ammonium acetate and 0.1 vol% of formic acid; the mobile phase B is acetonitrile; the flow rate of the mobile phases A and B is 0.2 mL/min;
the elution mode is gradient elution, and the procedure of the gradient elution is as follows:
when 0-3 min is reached, the volume percentage content of the mobile phase B is 20-60%;
When the time is 3-4 min, the volume percentage content of the mobile phase B is 60-90%;
when the time is 4-4.5 min, the volume percentage content of the mobile phase B is 90-20%;
4.5-7. min, wherein the volume percentage of the mobile phase B is 20%.
Preferably, the detection conditions of mass spectrometry include:
the mass spectrometer is a triple quadrupole mass spectrometer and adopts an ESI ionization source, a positive ion mode and a multi-reaction monitoring MRM mode;
the temperature of the drying gas is 3250 ℃; the flow rate of the drying gas is 10L/min; the atomization gas pressure was 20 psi; the capillary voltage was 4000V.
The invention provides a method for detecting phenylbutazone in horse meat based on a eutectic solvent, wherein an ammonium acetate buffer solution containing ascorbic acid is used as an extraction solvent, the acidic ammonium acetate buffer solution can increase the solubility of the phenylbutazone and improve the extraction efficiency of liquid-liquid microextraction, and the ascorbic acid contained in an extraction solution can avoid the decomposition phenomenon of the phenylbutazone in an acidic solution; meanwhile, the invention uses the ammonium acetate buffer solution containing ascorbic acid as the extraction solvent, thus avoiding the use of a large amount of organic extraction solvent. The invention uses the eutectic solvent as the extractant of liquid-liquid microextraction, can be mutually dissolved with the extraction solvent and quickly reach extraction balance, thereby improving the extraction efficiency. The method can realize the layering of the extracting agent and the extracting solvent by using the polar organic dispersing agent, and has simple operation and short time consumption. The results of the examples show that the detection method provided by the invention has good repeatability and precision.
Drawings
FIG. 1 is a chromatogram of a test solution in example 1;
FIG. 2 is a standard graph obtained in example 1;
FIG. 3 is a chromatogram of the standard solution in example 1;
FIG. 4 is a bar graph showing the recovery rates of eutectic solvents in solutions to be tested in examples 1 to 4;
FIG. 5 is a bar graph showing the recovery rates of eutectic solvents in solutions to be tested in examples 1 and 5 to 7;
FIG. 6 is a horse meat negative sample chromatogram;
FIG. 7 is a standard addition chromatogram of horse meat at a level of 10. mu.g/kg.
Detailed Description
The invention provides a method for detecting phenylbutazone in horse meat based on a eutectic solvent, which comprises the following steps:
(1) mixing a horse meat sample with an extraction solvent, a water-soluble inorganic salt, a eutectic solvent and a polar organic dispersant, sequentially carrying out liquid-liquid microextraction and centrifugation, taking supernatant liquor to a constant volume, and obtaining a liquid to be detected after the supernatant liquor passes through a membrane;
the extraction solvent is an ammonium acetate buffer solution containing ascorbic acid; the pH value of the ammonium acetate buffer solution containing ascorbic acid is 3-5;
the hydrogen bond acceptor of the eutectic solvent is choline chloride, and the hydrogen bond donor is phenol;
(2) detecting the liquid to be detected by using a high performance liquid chromatography-tandem mass spectrometry method to obtain the peak area of the liquid to be detected;
(3) Obtaining the content of phenylbutazone in horse meat according to the standard curve and the peak area obtained in the step (2); the standard curve is a linear relation curve of the concentration of the phenylbutazone standard solution and the peak area of the high performance liquid chromatography-tandem mass spectrum.
The horse meat sample is mixed with an extraction solvent, a water-soluble inorganic salt, a eutectic solvent and a polar organic dispersant, liquid-liquid microextraction and centrifugation are sequentially carried out, supernatant liquor is taken for constant volume, and a liquid to be detected is obtained after the supernatant liquor passes through a membrane. In the present invention, the horse meat sample is preferably a comminuted horse meat sample; the present invention does not require any particular way of comminution, as is well known to those skilled in the art.
In the invention, the extraction solvent is ammonium acetate buffer solution containing ascorbic acid; the pH value of the ammonium acetate buffer solution containing the ascorbic acid is 3-5, and the pH value is preferably 3.5. In the invention, the ammonium acetate buffer solution containing ascorbic acid is preferably an ammonium acetate aqueous solution containing ascorbic acid, and the molar concentration of ascorbic acid in the ammonium acetate buffer solution containing ascorbic acid is preferably 0.005-0.05 mol/L, and more preferably 0.01 mol/L; the molar concentration of the ammonium acetate is preferably 20 mmol/L. According to the method, an ammonium acetate buffer solution containing ascorbic acid is used as an extraction solvent, the acidic ammonium acetate buffer solution can increase the solubility of the phenylbutazone and improve the extraction efficiency of liquid-liquid microextraction, and the ascorbic acid contained in an extracting solution can avoid the decomposition phenomenon of the phenylbutazone in an acidic solution; meanwhile, the invention uses the ammonium acetate buffer solution containing ascorbic acid as the extraction solvent, thus avoiding the use of a large amount of organic extraction solvent.
In the present invention, the preparation method of the extraction solvent preferably comprises the steps of:
adjusting the pH value of the ammonium acetate aqueous solution to 3-5, and adding ascorbic acid for mixing to obtain an extraction solvent.
In the present invention, the pH adjusting agent is preferably acetic acid; the present invention does not require any particular mixing means, and mixing means known to those skilled in the art may be used.
In the invention, the hydrogen bond acceptor of the eutectic solvent is choline chloride, the hydrogen bond donor is phenol, and the molar ratio of the choline chloride to the phenol in the eutectic solvent is preferably 1: 1-4, and more preferably 1: 2. In the invention, the eutectic solvent is used as an extractant for liquid-liquid microextraction, and can be mutually dissolved with the extraction solvent and quickly reach extraction balance, thereby improving the extraction efficiency. In the present invention, the preparation method of the eutectic solvent preferably includes the steps of:
choline chloride and phenol were mixed to obtain a eutectic solvent.
The present invention preferably performs the mixing at normal temperature; in the invention, the mixing mode is preferably vortex mixing, the rotating speed of the vortex mixing is preferably 2000r/min, and the time is preferably 5 min.
In the present invention, the water-soluble inorganic salt is preferably sodium chloride, and the use of the water-soluble inorganic salt can change the ionic strength of the extract solution to produce a salting-out effect, thereby improving the extraction efficiency.
In the present invention, the polar organic dispersant is preferably one of acetonitrile, acetone, and tetrahydrofuran, and more preferably acetonitrile. The invention can lead the extractant and the extraction solvent to be layered quickly by using the polar organic dispersant.
In the present invention, the horse meat sample, the extraction solvent, the water-soluble inorganic salt, the eutectic solvent and the polar organic dispersant are preferably used in an amount ratio of 2.5 g: 5mL of: 1.0 g: 0.8 mL: 0.8 mL. In the mixing process, preferably, a horse meat sample and an extraction solvent are firstly mixed to obtain a first mixed solution; the first mixing mode is preferably shaking mixing, and the mixing time is preferably 10 min. After the first mixing is finished, adding water-soluble inorganic salt and a eutectic solvent into the first mixed solution for second mixing to obtain a second mixed solution; the second mixing mode is preferably vortex mixing, and the mixing time is preferably 5 min. After the second mixing is completed, the invention adds a polar organic dispersant to the second mixed solution.
In the present invention, the time of the liquid-liquid microextraction is preferably 5 min; the liquid-liquid micro-extraction is preferably carried out under the condition of vortex, and the rotating speed of the vortex is preferably 2000 r/min. In the present invention, the rotation speed of the centrifugation is preferably 10000r/min, and the time is preferably 5 min.
In the present invention, the solvent for constant volume is preferably the same as the polar organic dispersant. The invention has no special requirement on the volume fixing mode, and the volume fixing mode known by the technical personnel in the field can be used. In the invention, the mass ratio of the volume of the liquid to be measured after constant volume to the horse meat sample is preferably 1mL:2.5 g. The invention has no special requirement on the membrane passing mode, and the membrane passing mode known to the technical personnel in the field can be used.
After the liquid to be detected is obtained, the liquid to be detected is detected by using a high performance liquid chromatography-tandem mass spectrometry method, so that the peak area of the liquid to be detected is obtained. In the present invention, the detection conditions of the high performance liquid chromatography preferably include:
the chromatographic column is Acquity UPLC BEH C18, the length of the chromatographic column is 50mm, the diameter of the chromatographic column is 2.1mm, and the particle size of the filler is 1.7 μm;
the sample injection amount is 5 mu L;
mobile phase a was an aqueous solution containing 5mmol ammonium acetate and 0.1 vol% formic acid; the mobile phase B is acetonitrile; the flow rate of the mobile phases A and B is 0.2 mL/min;
The elution mode is gradient elution, and the procedure of the gradient elution is as follows:
when 0-3 min is reached, the volume percentage content of the mobile phase B is 20-60%;
when the time is 3-4 min, the volume percentage content of the mobile phase B is 60-90%;
when the time is 4-4.5 min, the volume percentage content of the mobile phase B is 90-20%;
4.5-7. min, wherein the volume percentage of the mobile phase B is 20%.
In the present invention, the detection conditions for mass spectrometry preferably include:
the mass spectrometer is a triple quadrupole mass spectrometer and adopts an ESI ionization source, a positive ion mode and a multi-reaction monitoring MRM mode;
the temperature of the drying gas is 3250 ℃; the flow rate of the drying gas is 10L/min; the atomization gas pressure was 20 psi; the capillary voltage was 4000V.
After the peak area is obtained, the content of phenylbutazone in horse meat is obtained according to a standard curve and the peak area; the standard curve is a linear relation curve of the concentration of the phenylbutazone standard solution and the peak area of the high performance liquid chromatography-tandem mass spectrum. In the present invention, the method for obtaining the standard curve preferably includes the steps of:
(a) providing a phenylbutazone standard solution;
(b) and detecting the phenylbutazone standard solution by using a high performance liquid chromatography-tandem mass spectrometry method, and drawing a standard curve by taking the concentration of the phenylbutazone standard solution as an abscissa and taking a peak area as an ordinate.
In the present invention, the concentration of the phenylbutazone standard solution is preferably 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, and 500ng/mL, and the solvent of the phenylbutazone standard solution is preferably acetonitrile. The preparation method of the standard solution of phenylbutazone in the invention has no special requirements, and the preparation method of the standard solution well known to those skilled in the art can be used.
In the invention, when the phenylbutazone standard solution is detected by using the high performance liquid chromatography-tandem mass spectrometry, the detection conditions of the high performance liquid chromatography and the detection conditions of the mass spectrometry are the same as those of the above, and are not repeated herein.
The method for drawing the standard curve has no special requirements, and the standard curve drawing method known to a person skilled in the art can be used. As an embodiment of the present invention, the standard curve is Y-83.3558X-657.9051, and the correlation coefficient R is2＝0.9991。
After the standard curve is obtained, the content of phenylbutazone in horse meat is obtained according to the standard curve and the peak area obtained in the step (2). In the present invention, the content of phenylbutazone in a horse meat sample is calculated according to formula I:
x is C V/m, formula I;
in formula I:
X is the content of the phenylbutazone in the horse meat sample, and the unit is mu g/kg;
c is the concentration of the phenylbutazone in the solution to be detected, and is calculated according to a standard curve, wherein the unit is mu g/L;
v is the constant volume, and the unit is mL;
and m is the mass of the horse meat sample to be detected, and the unit is g.
In the invention, the detection limit of the phenylbutazone in the horse meat sample is 5.0 mu g/kg.
The method for detecting phenylbutazone in horse meat based on eutectic solvent provided by the present invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Preparation of extraction solvent ammonium acetate buffer containing ascorbic acid: adding 20mmol/L ammonium acetate aqueous solution, adjusting pH to 3.5 with acetic acid, and adding ascorbic acid with ascorbic acid concentration of 0.01 mol/L.
Preparation of eutectic solvent: choline chloride is taken as a hydrogen bond as an acceptor, phenol is taken as a hydrogen bond donor, the choline chloride and the sodium hydrogen carbonate are mixed at normal temperature, and vortex is carried out at the speed of 2000r/min to obtain transparent clear liquid; the molar ratio of choline chloride to phenol was 1: 2.
(2) Weighing 2.5g of uniformly stirred horse meat sample to be detected, placing the horse meat sample to be detected in a 10mL plastic centrifuge tube with a plug, adding 5mL of the extraction solvent, uniformly mixing and shaking for 10min, adding 1.0g of sodium chloride, adding 0.8mL of the eutectic solvent as an extracting agent, uniformly mixing, adding 0.8mL of dispersing agent acetonitrile, uniformly mixing by vortex at a speed of 2000r/min for 5min, centrifuging at a speed of 10000r/min for 5min, taking supernatant, fixing the volume to 1.0mL by using acetonitrile, and passing through a membrane to obtain liquid to be detected.
Detecting the liquid to be detected by using a high performance liquid chromatography-tandem mass spectrometry instrument, wherein the high performance liquid chromatography parameters are as follows: the chromatographic column is AcquisyUPLC BEH C18, the column length of the chromatographic column is 50mm, the column diameter is 2.1mm, and the particle size of the filler is 1.7 mu m;
the sample injection amount is 5 mu L;
mobile phase a was an aqueous solution containing 5mmol ammonium acetate and 0.1 vol% formic acid; the mobile phase B is acetonitrile; the flow rate of the mobile phases A and B is 0.2 mL/min;
the elution mode is gradient elution, and the procedure of the gradient elution is as follows:
when 0-3 min is reached, the volume percentage content of the mobile phase B is 20-60%;
when the time is 3-4 min, the volume percentage of the mobile phase B is 60-90% B;
when the time is 4-4.5 min, the volume percentage of the mobile phase B is 90-20% B;
4.5-7. min, wherein the volume percentage of the mobile phase B is 20%;
the mass spectrum parameters are as follows: the mass spectrometer is a triple quadrupole mass spectrometer and adopts an ESI ionization source, a positive ion mode and a multi-reaction monitoring MRM mode;
the temperature of the drying gas is 3250 ℃; the flow rate of the drying gas is 10L/min; the atomization gas pressure was 20 psi; the capillary voltage was 4000V. In mass spectrometry, the ion pair of phenylbutazone MRM is 309/188 (quantitative) and 309/160, and the chromatogram of the obtained solution to be detected is shown in FIG. 1, and the peak area is 1010.
(3) Using acetonitrile as a solvent to prepare 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL phenylbutazone standard solutions, detecting the phenylbutazone standard solutions by using the high performance liquid chromatography-tandem mass spectrometry, drawing a standard curve by using the concentration of the phenylbutazone standard solutions as an abscissa and using peak areas as an ordinate, wherein the obtained standard curve is shown in FIG. 2, and the result shows that the linearity of the phenylbutazone in the range of 10-500 ng/mL is good, the regression equation data is shown in Table 1, and the chromatogram of the 20ng/mL phenylbutazone standard solution is shown in FIG. 3.
TABLE 1 Linear regression equation data
The method for qualitatively judging the phenylbutazone in the liquid to be detected comprises the following steps:
if the retention time of chromatographic peaks in the liquid to be detected is consistent with that of a standard solution, the relative abundance of two daughter ions of phenyl butyrone in the liquid to be detected is consistent with that of the standard solution with the same concentration, and the relative abundance deviation does not exceed the allowable relative deviation (the relative abundance is more than 50%, the deviation of +/-20%, the relative abundance is 20-50%, the deviation of +/-25%, the relative abundance is 10-20%, the deviation of +/-30%, and the relative abundance is less than 10%, the deviation of +/-50%) then the existence of phenyl butyrone in the sample can be judged.
As can be seen from comparison between fig. 1 and fig. 3, the retention time of the chromatographic peak in the liquid to be tested is consistent with that of the standard solution, and both retention times are 4.88min, and the relative abundance of two daughter ions of phenylbutazone in the liquid to be tested is consistent with that of the standard solution with the same concentration, and the deviation of the relative abundance is 10.6%, which indicates that phenylbutazone exists in the horse meat sample of the present embodiment.
And quantitatively detecting the content of the phenylbutazone in the liquid to be detected, and calculating the content of the phenylbutazone in the horse meat sample to be 8.0 mu g/kg according to the figure 1 and a standard curve.
Examples 2 to 4
Example 2 differs from example 1 in that the molar ratio of choline chloride to phenol in the eutectic solvent is 1: 1; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 8.2 mug/kg.
Example 3 differs from example 1 in that the molar ratio of choline chloride to phenol in the eutectic solvent is 1: 3; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 7.0 mu g/kg.
Example 4 differs from example 1 in that the molar ratio of choline chloride to phenol in the eutectic solvent is 1: 4; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 6.0 mu g/kg.
The recovery rate of phenylbutazone in the solutions tested in examples 1-4 is shown in FIG. 4. As can be seen from FIG. 4, the eutectic solvent used in the present invention has a higher recovery rate, and the recovery rate of phenylbutazone is higher when the molar ratio of choline chloride to phenol is 1:1 and 1: 2.
Examples 5 to 7
Example 5 differs from example 1 in that the amount of eutectic solvent used was 0.3 mL; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 6.3 mug/kg.
Example 6 differs from example 1 in that the amount of the eutectic solvent used was 0.5 mL; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 7.2 mug/kg.
Example 7 differs from example 1 in that the amount of the eutectic solvent used was 1 mL; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 8.3 mu g/kg.
The recovery rates of phenylbutazone in examples 1 and 5-7 are shown in FIG. 5. As can be seen from FIG. 5, the eutectic solvent used in the present invention has a higher recovery rate, and the recovery rate of phenylbutazone is higher when the amount of the eutectic solvent is 0.8mL and 1 mL.
Examples 8 to 9
Example 8 differs from example 1 in that the dispersant is acetone; the quantitative detection of the phenylbutazone in the liquid to be detected is carried out according to the method in the embodiment 1, and the detection result shows that the content of the phenylbutazone in the horse meat sample is 3.3 mug/kg.
Example 9 differs from example 1 in that the dispersant is tetrahydrofuran; the quantitative determination of the phenylbutazone in the solution to be determined was carried out according to the method of example 1, and the determination result was that the content of phenylbutazone in the horse meat sample was 4.9. mu.g/kg.
Test example 1 verification of precision
Horse meat samples (i.e., horse meat negative samples) without phenylbutazone were tested according to the method of example 1, and the chromatogram of the obtained horse meat negative samples is shown in FIG. 6.
3 parts of 2.5g of uniformly stirred horse meat negative samples are weighed, 5.0 mu g/kg, 10 mu g/kg and 50 mu g/kg levels of phenylbutazone standard substances (6 parallel samples are arranged at each addition level) are respectively added to serve as horse meat standard addition samples, the phenylbutazone in the horse meat standard addition samples is quantitatively detected according to the method of example 1, and the average recovery rate and the relative standard deviation are calculated, and the results are shown in table 2. The standard addition chromatogram of horse meat at a 10. mu.g/kg level is shown in FIG. 7.
TABLE 2 average recovery and relative standard deviation test results
As can be seen from Table 2, the detection method for phenylbutazone in horse meat based on the eutectic solvent provided by the invention is good in repeatability and precision.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for detecting phenylbutazone in horse meat based on a eutectic solvent comprises the following steps:
(1) mixing a horse meat sample with an extraction solvent, a water-soluble inorganic salt, a eutectic solvent and a polar organic dispersant, sequentially carrying out liquid-liquid microextraction and centrifugation, taking supernatant liquor to a constant volume, and obtaining a liquid to be detected after the supernatant liquor passes through a membrane;
the extraction solvent is an ammonium acetate buffer solution containing ascorbic acid; the pH value of the ammonium acetate buffer solution containing ascorbic acid is 3-5;
the hydrogen bond acceptor of the eutectic solvent is choline chloride, and the hydrogen bond donor is phenol;
(2) detecting the liquid to be detected by using a high performance liquid chromatography-tandem mass spectrometry method to obtain the peak area of the liquid to be detected;
(3) Obtaining the content of phenylbutazone in horse meat according to the standard curve and the peak area obtained in the step (2); the standard curve is a linear relation curve of the concentration of the phenylbutazone standard solution and the peak area of the high performance liquid chromatography-tandem mass spectrum.
2. The detection method according to claim 1, wherein the molar concentration of ascorbic acid in the ascorbic acid-containing ammonium acetate buffer solution is 0.005 to 0.05 mol/L, and the molar concentration of ammonium acetate is 20 mmol/L.
3. The detection method according to claim 1, wherein the molar ratio of choline chloride to phenol in the eutectic solvent is 1: 1-4.
4. The detection method according to claim 1, wherein the water-soluble inorganic salt is sodium chloride, and the polar organic dispersant is one of acetonitrile, acetone, and tetrahydrofuran.
5. The detection method according to claim 1 or 4, wherein the horse meat sample, the extraction solvent, the water-soluble inorganic salt, the eutectic solvent and the polar organic dispersant are used in an amount ratio of 2.5 g: 5mL of: 1.0 g: 0.8 mL: 0.8 mL.
6. The detection method according to claim 1, wherein the time for the liquid-liquid microextraction is 5 min; the rotation speed of the centrifugation is 10000r/min, and the time is 5 min.
7. The detection method according to claim 1, wherein the detection conditions of the high performance liquid chromatography comprise:
the chromatographic column is Acquity UPLC BEH C18, the length of the chromatographic column is 50mm, the diameter of the chromatographic column is 2.1mm, and the particle size of the filler is 1.7 mu m;
the sample injection amount is 5 mu L;
mobile phase a was an aqueous solution containing 5mmol/L ammonium acetate and 0.1 vol% formic acid; the mobile phase B is acetonitrile; the flow rate of the mobile phases A and B is 0.2 mL/min;
the elution mode is gradient elution, and the procedure of the gradient elution is as follows:
when 0-3 min is reached, the volume percentage content of the mobile phase B is 20-60%;
when the time is 3-4 min, the volume percentage content of the mobile phase B is 60-90%;
when the time is 4-4.5 min, the volume percentage content of the mobile phase B is 90-20%;
4.5-7 min, wherein the volume percentage of the mobile phase B is 20%.
8. The detection method according to claim 1, wherein the detection conditions of mass spectrometry include:
the mass spectrometer is a triple quadrupole mass spectrometer and adopts an ESI ionization source, a positive ion mode and a multi-reaction monitoring MRM mode;
the temperature of the drying gas is 3250 ℃; the flow rate of the drying gas is 10L/min; the atomization gas pressure was 20 psi; the capillary voltage was 4000V.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6368618B1 (en) * | 1999-07-01 | 2002-04-09 | The University Of Georgia Research Foundation, Inc. | Composition and method for enhanced transdermal absorption of nonsteroidal anti-inflammatory drugs |
| CN104965035A (en) * | 2015-04-27 | 2015-10-07 | 公安部物证鉴定中心 | Method for screening toxic substances in sample by using solid phase support liquid-liquid extraction-GC MS |
| CN107167539A (en) * | 2017-06-27 | 2017-09-15 | 中山出入境检验检疫局检验检疫技术中心 | The detection method of a variety of residues of veterinary drug in a kind of rapid screening flesh of fish |
| CN107255686A (en) * | 2017-08-14 | 2017-10-17 | 中山出入境检验检疫局检验检疫技术中心 | The analysis method of many residues of veterinary drug in a kind of measure cultivation water |
| CN107817306A (en) * | 2017-10-27 | 2018-03-20 | 中山出入境检验检疫局检验检疫技术中心 | Method that is a kind of while detecting 64 kinds of residues of veterinary drug in aquatic products |
| CN109115898A (en) * | 2018-06-14 | 2019-01-01 | 浙江工业大学 | Based on the remaining method of pesticide in eutectic solvent-inorganic salts double-aqueous phase system analysis water-like |
| CN109633032A (en) * | 2019-01-25 | 2019-04-16 | 云南省农业科学院农产品加工研究所 | Method that is a kind of while measuring malachite green and crystal violet content |
| EP3485874A1 (en) * | 2017-11-17 | 2019-05-22 | SeraNovo B.V. | Method of preparing a natural deep eutectic solvent (nades)-active ingredient system; a nades-, and preferably a polymeric precipitation inhibitor containing nades-, based drug formulation technology and development method |
| CN110231423A (en) * | 2019-07-05 | 2019-09-13 | 山西农业大学 | The method of pyrethroid pesticide in dispersive liquid-liquid microextraction-high performance liquid chromatography measurement grain |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018204424A1 (en) * | 2017-05-01 | 2018-11-08 | National Technology & Engineering Solutions Of Sandia, Llc | Novel compositions and methods for synthesizing deep eutectic solvents from lignin derived phenolic compounds |
-
2020
- 2020-07-03 CN CN202010635894.7A patent/CN111735883B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6368618B1 (en) * | 1999-07-01 | 2002-04-09 | The University Of Georgia Research Foundation, Inc. | Composition and method for enhanced transdermal absorption of nonsteroidal anti-inflammatory drugs |
| CN104965035A (en) * | 2015-04-27 | 2015-10-07 | 公安部物证鉴定中心 | Method for screening toxic substances in sample by using solid phase support liquid-liquid extraction-GC MS |
| CN107167539A (en) * | 2017-06-27 | 2017-09-15 | 中山出入境检验检疫局检验检疫技术中心 | The detection method of a variety of residues of veterinary drug in a kind of rapid screening flesh of fish |
| CN107255686A (en) * | 2017-08-14 | 2017-10-17 | 中山出入境检验检疫局检验检疫技术中心 | The analysis method of many residues of veterinary drug in a kind of measure cultivation water |
| CN107817306A (en) * | 2017-10-27 | 2018-03-20 | 中山出入境检验检疫局检验检疫技术中心 | Method that is a kind of while detecting 64 kinds of residues of veterinary drug in aquatic products |
| EP3485874A1 (en) * | 2017-11-17 | 2019-05-22 | SeraNovo B.V. | Method of preparing a natural deep eutectic solvent (nades)-active ingredient system; a nades-, and preferably a polymeric precipitation inhibitor containing nades-, based drug formulation technology and development method |
| CN109115898A (en) * | 2018-06-14 | 2019-01-01 | 浙江工业大学 | Based on the remaining method of pesticide in eutectic solvent-inorganic salts double-aqueous phase system analysis water-like |
| CN109633032A (en) * | 2019-01-25 | 2019-04-16 | 云南省农业科学院农产品加工研究所 | Method that is a kind of while measuring malachite green and crystal violet content |
| CN110231423A (en) * | 2019-07-05 | 2019-09-13 | 山西农业大学 | The method of pyrethroid pesticide in dispersive liquid-liquid microextraction-high performance liquid chromatography measurement grain |
Non-Patent Citations (2)
| Title |
|---|
| Recent developments in synthetic chemistry and biological activities of pyrazole derivatives;MUHAMMAD FAISAL et al.;《J. Chem. Sci.》;20191231;全文 * |
| 分散固相萃取－超高效液相色谱－串联质谱法测定奶粉中7 种非选择性环氧化酶抑制药物;励炯 等;《色谱》;20190228;全文 * |
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