CN112305127A - LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for sodium pentachlorophenate residue in eggs - Google Patents
LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for sodium pentachlorophenate residue in eggs Download PDFInfo
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Abstract
The invention discloses an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for sodium pentachlorophenate residue in eggs. The invention establishes a sample pretreatment method capable of effectively avoiding matrix interference in a sample, can be used for qualitative and quantitative detection of sodium pentachlorophenate in eggs, and has the advantages of average detection recovery rate of 78.99-81.92%, average Relative Standard Deviation (RSD) of 0.18-0.20%, detection limit of 0.5 mu g/kg and quantitative limit of 1 mu g/kg.
Description
Technical Field
The invention relates to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for sodium pentachlorophenate residue in eggs, belonging to the technical field of veterinary drug residue detection.
Background
The eggs are rich in protein and amino acid, and have high nutritive value. It is easy to be absorbed by human body, and has high utilization rate, and the amino acid proportion is suitable for human physiological requirement, and is one of the foods for human being. However, in recent years, food safety incidents that the residual medicines in eggs exceed standards frequently occur, and people worry about the quality safety of eggs.
Sodium pentachlorophenate is sodium salt of pentachlorophenol, belongs to high-toxicity organic chlorine pesticide, is commonly used as herbicide, wood preservative, antibacterial agent, pesticide and the like, and is used as a molluscicide in southern schistosomiasis epidemic areas in China in a large amount. Sodium pentachlorophenate has good water solubility, and can be widely spread through water carriers, enter the food chain and produce biological accumulation. The pentachlorophenol is easy to be converted into pentachlorophenol under an acid environment, has stable chemical properties, is difficult to degrade, and is easy to react with cells and functional proteins in blood when entering high-grade animals, so that the normal physiological activities of organs and tissues such as liver, kidney, central nervous system and the like are influenced. A large amount of residues can be enriched through a food chain, and the animal-derived food with the drug residues can cause carcinogenesis, teratogenesis, mutagenesis and other damages to human bodies after the human bodies eat the animal-derived food for a long time. The ministry of agriculture announcements No. 193 and No. 235 stipulate that pentachlorophenol and its sodium salt are prohibited from being used in all animal-derived foods, and are not detectable in animal-derived foods. The detailed implementation of national food safety supervision (2018 edition) also explicitly stipulates the items of pentachlorophenol and sodium salt thereof in livestock and poultry meat and byproducts thereof.
In conclusion, the detection of sodium pentachlorophenate residue in eggs is enhanced, so that the detection can be completely and effectively realizedThe control of the medicine really realizes the aims of guaranteeing food safety and protecting human health. At present, the detection methods of the medicines mainly comprise gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry and the like. The sodium pentachlorophenol can be completely converted into the pentachlorophenol under the acidic condition of pH 6.6-6.8, the pentachlorophenol is very stable, and the sodium pentachlorophenol is generally detected by firstly converting into the pentachlorophenol. The conjugated structure of the pentachlorophenol is stable, and the characteristic ion, namely pentachlorophenol ion [ M-H ], is difficult to obtain]-And is not easy to be broken in the triple quadrupole collision chamber. It has 5 chlorine atoms, exists35Cl and37cl isotope, so 4 isotope parent ion pairs (262.8/262.8, 264.8/264.8, 266.8/266.8,268.8/268.8) are adopted as qualitative and quantitative ion pairs to obtain 4 identification points so as to meet the confirmation requirement.
The method comprises the steps of firstly acidifying a sample by concentrated sulfuric acid to completely convert sodium pentachlorophenate into pentachlorophenol, extracting n-hexane, then sulfonating and purifying by concentrated sulfuric acid, and then measuring by LC-MS/MS. The linear range of the method is 0.5-200 mug/kg, the detection limit is 0.2 mug/kg, the correlation coefficient is 0.9993, and the sample standard adding recovery rate is 80.3-91.7%. The method comprises the steps of extracting a sample by using 8% triethylamine acetonitrile and water (80:20, V: V), purifying by using an EMR-Lipid solid phase extraction column, taking 5mmol/L ammonium acetate solution (containing 0.1% formic acid) -methanol as a mobile phase, performing chromatographic separation by using Waters acquisition BEH C18 (50mm multiplied by 2.1mm,1.7 mu M), wherein the pentachlorophenol is an acidic compound and is easy to lose hydrogen protons to obtain molecular ions, therefore, a negative ion mode [ M-H ] -can be used for obtaining higher sensitivity, the matrix is matched with an external standard method for quantification, and the result shows that the linearity of the pentachlorophenol in the range of 0.5-10 ng/mL is good, the detection limit of the method is 0.1-0.3 mu g/kg, the limit of quantification is 0.3-0.5 mug/kg, and the recovery rate of 6 matrixes is 81-98% under the concentration levels of 1.0, 5.0 and 10.0 mug/kg.
Therefore, most of the existing detection of sodium pentachlorophenate residues relates to chicken, tissues, feed and aquatic products, and few HPLC-MS/MS methods for detecting the sodium pentachlorophenate residues in eggs are available. Therefore, it is especially necessary to establish an effective, convenient and widely-applied liquid chromatography-tandem mass spectrometry method for detecting sodium pentachlorophenate in eggs.
Disclosure of Invention
The invention aims to provide an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for the residual amount of sodium pentachlorophenate in eggs, which is mainly used for determining the residual amount of sodium pentachlorophenate in eggs.
The above purpose is realized by the following technical scheme:
an LC-MS/MS determination method for the residual amount of sodium pentachlorophenate in eggs comprises the following steps:
(1) extraction of
Homogenizing and stirring eggs uniformly, weighing 2g of sample in a centrifuge tube, adding 2mL of water, homogenizing for 1min, adding 5mL of 2-10% (volume) ammonia acetonitrile solution, firstly whirling for 30 seconds, then carrying out ultrasonic extraction for 10min, then centrifuging for 8min at 4 ℃ at 10000r/min, transferring supernatant, repeating the extraction step for 1 time by using 5mL of 2-10% ammonia acetonitrile solution on residues, and combining the two supernatants;
(2) purification
Eluting the solid-phase extraction column with 3mL of methanol and 3mL of water in sequence for activation, then completely loading the supernatant obtained in the step (1) onto the solid-phase extraction column, discarding effluent liquid, eluting with 5mL of 5 vol% ammonia water solution, 5mL of methanol and 5mL of 2 vol% methanol-formic acid-water solution (obtained by mixing 4 vol% methanol formate solution and water in equal volumes) in sequence, discarding effluent liquid after eluent liquid completely passes through, draining the solid-phase extraction column with a vacuum pump, eluting with 3mL of 8 vol% methanol formate solution, collecting eluent into a clean test tube, adding water to accurately fix the volume to 4mL, mixing uniformly, and allowing the solution to pass through an Agilent PTFE-Q syringe filter to obtain a sample solution to be detected;
(3) preparation of Standard solutions
Diluting the pentachlorophenol standard substance with an initial mobile phase to prepare a standard substance solution with the pentachlorophenol concentration of 1, 2, 5, 10 and 20 mu g/kg;
(4) measurement and calculation of results
Respectively carrying out LC-MS/MS measurement on the sample solution to be measured and the standard solution under the same conditions, wherein the measurement conditions are as follows:
liquid chromatography conditions:
a chromatographic column: c18
Mobile phase: the phase A is 2-10mmol/L ammonium acetate solution; phase B is methanol solution
Column temperature: 25 deg.C
Sample introduction amount: 20 μ L
Gradient elution:
| time/min | A/% (volume) | B/% (volume) |
| 0.0 | 60 | 40 |
| 7.0 | 0 | 100 |
| 7.5 | 60 | 40 |
| 12 | 60 | 40 |
Mass spectrum conditions:
the system comprises the following steps: agilent 6470 triple quadrupole LC-MS (liquid chromatography-mass spectrometry) system
An ion source: ESI-
Temperature of the drying gas: 300 deg.C
Flow rate of the dryer: 5L/min
Atomizer pressure: 45psi
Temperature of sheath gas: 250 deg.C
Flow rate of sheath gas: 11L/min
Scanning mode: MRM
Capillary voltage: 3500V (-)
Nozzle voltage: 500V (-)
Ion pairs monitored in multiple reaction monitoring mode:
and drawing a standard curve by taking the peak area of the standard substance solution characteristic ion mass chromatogram as the ordinate and the concentration as the abscissa. Substituting the chromatographic peak area of the pentachlorophenol in the sample solution to be detected into a standard curve to obtain the pentachlorophenol content in the sample solution to be detected and calculating the residual amount of sodium pentachlorophenol in the eggs.
Preferably, the solid phase extraction column is an Agilent Bond Elut Plexa PAX solid phase extraction cartridge.
Preferably, the concentration of the ammonia acetonitrile solution in the step (1) is 5%.
Preferably, the column is Agilent ZORBAX Eclipse Plus C18, 2.1X 50mm,1.8 μm in size.
Preferably, the A phase is a 5mmol/L ammonium acetate solution.
The invention has the beneficial effects that:
the invention establishes a sample pretreatment method which is simple, convenient and rapid and can effectively avoid matrix interference in a sample by optimizing an extraction solvent, a solid-phase extraction condition, a chromatographic column and a mobile phase, selecting a filter membrane and applying LC-MS/MS to qualitative and quantitative detection of sodium pentachlorophenate in eggs. Experiments show that the method has good extraction and purification effects by using a 5% ammonia water-acetonitrile extraction column, an Agilent Bond Elut Plexa PAX solid phase extraction column, a gradient elution program using Agilent ZORBAX Eclipse Plus C18 (2.1X 50mm,1.8 mu m) chromatographic column and 5mmol/L ammonium acetate solution-methanol as a mobile phase, good peak shape and high recovery rate, the average recovery rate is 78.99-81.92%, the average Relative Standard Deviation (RSD) is 0.18-0.20%, the detection limit is 0.5 mu g/kg, the quantification limit is 1 mu g/kg, and the method has the advantages of simplicity and convenience in operation, rapidness, accuracy, high sensitivity and good repeatability.
Drawings
FIG. 1: the working curve of the pentachlorophenol standard and the matrix match the working curve.
FIG. 2: chromatogram of pentachlorophenol in egg (1. mu.g/kg)
FIG. 3: blank egg chromatogram.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Instruments and reagents used in the examples
Agilent 6470 triple quadrupole LC-MS system with distributed spray ion source; analytical balance: 0.00001g of sensory quantity and 0.001g of sensory quantity; volumetric flask: 100mL and 200 mL; 1mL of pipette, 100. mu.L of pipette; freezing a high-speed centrifuge (10000 r/min); a constant-temperature water bath kettle; a vortex mixer; a solid phase extraction device; an ultrasonic cleaning machine; solid phase extraction column: agilent Bond Elut Plexa PAX,60mg,3 mL; and (3) filtering the membrane: agilent PTFE-Q syringe needle filter, 0.2 μm, 13 mm; and (4) a common filter membrane.
Acetonitrile and methanol are in chromatographic purity. Acetic acid, formic acid, ammonia, and the like are available from the national institutes. Sodium pentachlorophenol with a purity of 86.0% was purchased from dr. ehrensorfer; pentachlorophenol standard solution (0.998mg/mL in methanol) was purchased from the institute of veterinary medicine, China; the egg samples were all commercially available products. All reagents were analytically pure except as otherwise specified, and water was first grade water in accordance with the GB/T6682 specification.
Example 1: assay method and condition optimization
(1) Selection of extraction solvent
Taking a sample to be tested, homogenizing and stirring uniformly, weighing 2 +/-0.02 g of sample in a 50mL centrifuge tube, observing the extraction effects of acetonitrile, 2% ammonia water-acetonitrile, 5% ammonia water-acetonitrile and 8% ammonia water-acetonitrile, fully extracting with 5mL acetonitrile, 2% ammonia water-acetonitrile, 5% ammonia water-acetonitrile and 8% ammonia water-acetonitrile respectively, then vortexing for 30 seconds, ultrasonically extracting for 10min, centrifuging at 4 ℃ for 8min at 10000r/min, transferring a supernatant into another test tube, repeating the extraction step for 1 time by using 5mL 5% ammonia water-acetonitrile solution on the residue after centrifugation, and combining the supernatants. Finally, 5% ammonia water-acetonitrile is selected to have better extraction effect.
(2) Determination of solid phase extraction conditions
Selection of a solid phase extraction column: the extraction recovery of C18, Oasis HLB and Oasis MAX and Agilent Bond Elut Plexa PAX solid phase extraction columns were compared, and finally Agilent Bond Elut Plexa PAX solid phase extraction columns were selected.
Elution: after determining the use of the Agilent Bond Elut Plexa PAX, the Agilent Bond Elut Plexa PAX column was rinsed with 3mL of methanol followed by 3mL of water for activation. And (3) completely loading the supernatant obtained in the extraction step onto a Bond Elut Plexa PAX small column, discarding the effluent, sequentially eluting with 5mL of 5% ammonia water solution, 5mL of methanol and 5mL of 2% methanoic acid methanol-water solution, discarding the effluent after the eluent completely passes through the small column, pumping out the solid phase extraction column for 2min by using a vacuum pump, eluting with 3mL of 8% methanoic acid methanol solution, pumping out the small column, collecting the eluent into a clean 15mL graduated test tube, adding water, accurately metering the volume to 4mL, and uniformly mixing.
(3) Preparation of Standard solutions
Pentachlorophenol standard is diluted with an initial mobile phase to form standard solutions with the concentrations of 1, 2, 5, 10 and 20 mu g/kg, and the standard solutions are subjected to liquid chromatography-tandem mass spectrometry. And drawing a standard curve by taking the peak area of the characteristic ion mass chromatogram as a vertical coordinate and the concentration of the standard solution as a horizontal coordinate. And solving a regression equation and a correlation coefficient.
(4) Selection of the filter membrane: the literature reports that when filter membranes are used for filtration and impurity removal in the previous step of machine loading, common filter membranes have adsorption on pentachlorophenol, so that a large amount of pentachlorophenol compounds are lost, and the recovery rate is low, therefore, compared with the influence of different filter membranes on the recovery rate of pentachlorophenol, the eluent is filtered through 0.22 mu m and Agilent PTFE-Q filter membranes in sequence, and is analyzed by machine loading.
(5) Selection of chromatography columns and mobile phases
Two columns of ZORBAX Eclipse XDB-C18(1.8 μm, 50X 2.1mm) and Agilent ZORBAX Eclipse Plus C18 (2.1X 50mm,1.8 μm) were compared, the latter being finally selected.
Mobile phase: the research compares the mobile phase system of the combination of the A phase of 5mmol/L ammonium acetate solution and the B phase of 2moL/L ammonium acetate solution (containing 0.1 percent of formic acid), and finally selects the 5mmol/L ammonium acetate solution as the A phase, so that the separation effect is better.
(6) Preparation of matrix matching solution
And (3) diluting the standard stock solution by using a blank matrix solution (namely an egg sample solution without sodium pentachlorophenate), obtaining a standard matrix working solution with the pentachlorophenol concentration of 1, 2, 5, 10 and 20 mu g/kg, analyzing by using a high performance liquid chromatography-tandem mass spectrometer, quantifying the peak area of each component in a quantitative ion chromatogram, and drawing a matrix matching working curve.
(7) Measurement and calculation of results
Liquid chromatography conditions:
column temperature: 25 deg.C
Sample introduction amount: 20 μ L
Gradient elution: the gradient elution procedure is shown in Table 1
TABLE 1 gradient elution procedure
| Time/min | A/％ | B/％ |
| 0.0 | 60 | 40 |
| 7.0 | 0 | 100 |
| 7.5 | 60 | 40 |
| 12 | 60 | 40 |
Mass spectrum conditions:
the system comprises the following steps: agilent 6470 Triplex quadrupole LC-MS;
an ion source: ESI-;
temperature of the drying gas: 300 ℃;
flow rate of the dryer: 5L/min;
atomizer pressure: 45 psi;
temperature of sheath gas: 250 ℃;
flow rate of sheath gas: 11L/min;
scanning mode: MRM;
capillary voltage: 3500V (-);
nozzle voltage: 500V (-);
the ion pairs monitored in the multiple reaction monitoring mode are shown in table 2.
TABLE 2 ion pairs monitored by pentachlorophenol multiple reaction monitoring mode
And respectively loading the standard substance solution, the standard matrix working solution and the sample solution to be detected for detection.
Example 2 methodological examination
1. Qualitative determination
And comparing and determining the retention time of the sample chromatogram with the retention time of the standard substance, and comparing the characteristic ions of the chromatographic peak with the characteristic ions of the chromatographic peak of the standard substance with corresponding concentrations. The relative deviation of the retention time of the sample from the standard is not more than 2.5%; the relative abundance of the characteristic ions of the sample is consistent with that of the standard solution with the equivalent concentration, and the deviation of the relative abundance does not exceed the specification of the table 3, so that the corresponding detected object in the sample can be judged. The retention time, deviation within ± 5%, and the relative abundance of the detected ions should be consistent with the relative abundance of the calibration standard solution at comparable concentrations. The allowable deviation should meet the requirements of table 3.
TABLE 3 maximum permissible error in relative ion abundance for qualitative confirmation
| Relative ion abundance (%) | ＞50 | > 20 to 50 | > 10 to 20 | ≤10 |
| Maximum deviation allowed (%) | ±20 | ±25 | ±30 | ±50 |
2. Quantitative determination
Under the optimal working condition of the instrument, the matrix mixed standard working solution is injected, a standard working curve is drawn by taking the peak area as the ordinate and the concentration of the matrix mixed standard working solution as the abscissa, the sample is quantified by using the standard working curve, and the response value of the object to be measured in the sample solution is in the linear range measured by the instrument.
The establishment of the detection method comprises the following steps:
(1) standard curve
Taking the sample solution and the corresponding standard solution, carrying out single-point or multi-point calibration, and quantifying according to the peak area of an external standard method, wherein the response values of the pentachlorophenol in the standard solution and the sample solution are within the linear range detected by an instrument.
(2) Matrix standard curve
And (3) diluting the standard stock solution by using a blank matrix solution to obtain a standard matrix working solution with the pentachlorophenol concentration of 1, 2, 5, 10 and 20 mu g/kg, then carrying out high performance liquid chromatography-tandem mass spectrometer analysis, quantifying the peak area of each component in a quantitative ion chromatogram, drawing a matrix matching working curve, and obtaining that the matrix matching linearity of the sodium pentachlorophenol in the egg is 1-20 mu g/kg, and the correlation coefficient is more than 0.99. The working curve of the standard substance and the working curve of the matrix matching are shown in figure 1, the matching degree of the two is very high, which shows that the influence of the extracted and purified sample matrix on the detection result is very small, and the recovery rate can be directly calculated through the linear curve of the standard substance.
(3) Sensitivity of the probe
The reliability of the measurement result in the trace analysis depends on the size and fluctuation condition of the blank value to a great extent, let Wt represent the total value of the tested sample, Wb represent the blank value, the lower limit of the sample detection is the content (Wt-Wb) of the tested component is 3 sigma, the lower limit of the quantitative detection is 10 sigma, the lower limit of the actual detection by the method is 0.5 mug/kg, and the lower limit of the quantitative detection is 1 mug/kg.
(4) Accuracy and precision
Under the optimal test conditions, the samples of the egg are extracted and measured at different adding concentrations, the accuracy of the method is expressed by the recovery rate, and the precision is expressed by the relative deviation. Specifically, 1, 2 and 5 mug/kg of 3 concentration levels of sodium pentachlorophenate standard solutions are added into eggs without sodium pentachlorophenate, and the residual quantity is determined according to the processing steps. The measured concentrations were compared with the theoretical drug addition concentrations to obtain the drug addition recovery rates, and each addition level was measured in parallel 5 times to obtain the relative standard deviation, the measurement results are shown in table 4. As can be seen from Table 4, the average recovery rate of pentachlorophenol is 78.99% -81.92% and the average Relative Standard Deviation (RSD) is 0.18% -0.20% at 3 standard addition levels, which indicates that the method of the present invention has high recovery rate and good repeatability.
TABLE 4 recovery of pentachlorophenol in egg samples
Claims (5)
1. An LC-MS/MS method for determining the residual amount of sodium pentachlorophenate in eggs is characterized by comprising the following steps:
(1) extraction of
Homogenizing and stirring eggs uniformly, weighing 2g of sample in a centrifuge tube, adding 2mL of water, homogenizing for 1min, adding 5mL of 2-10% ammonia acetonitrile solution, firstly whirling for 30 seconds, then carrying out ultrasonic extraction for 10min, then centrifuging for 8min at 4 ℃ at 10000r/min, transferring supernatant, repeating the extraction step for 1 time by using 5mL of 2-10% ammonia acetonitrile solution on residues, and combining the two supernatants;
(2) purification
Eluting the solid-phase extraction column with 3mL of methanol and 3mL of water in sequence for activation, then completely loading all the supernatant obtained in the step (1) onto the solid-phase extraction column, discarding effluent liquid, eluting with 5mL of 5% ammonia water solution, 5mL of methanol and 5mL of 2% methanoic acid methanol-water solution in sequence, discarding the effluent liquid after the eluent completely passes through, pumping out the solid-phase extraction column by using a vacuum pump, eluting with 3mL of 8% methanoic acid methanol solution, collecting the eluent into a clean test tube, adding water to accurately fix the volume to 4mL, mixing uniformly, and enabling the solution to pass through an Agilent PTFE-Q syringe filter to obtain a sample solution to be detected;
(3) preparation of Standard solutions
Diluting the pentachlorophenol standard substance with an initial mobile phase to prepare a standard substance solution with the pentachlorophenol concentration of 1, 2, 5, 10 and 20 mu g/kg;
(4) measurement and calculation of results
Respectively carrying out LC-MS/MS measurement on the sample solution to be measured and the standard solution under the same conditions, wherein the measurement conditions are as follows:
liquid chromatography conditions:
a chromatographic column: c18
Mobile phase: the phase A is 2-10mmol/L ammonium acetate solution; phase B is methanol solution
Column temperature: 25 deg.C
Sample introduction amount: 20 μ L
Gradient elution:
mass spectrum conditions:
the system comprises the following steps: agilent 6470 triple quadrupole LC-MS (liquid chromatography-mass spectrometry) system
An ion source: ESI-
Temperature of the drying gas: 300 deg.C
Flow rate of the dryer: 5L/min
Atomizer pressure: 45psi
Temperature of sheath gas: 250 deg.C
Flow rate of sheath gas: 11L/min
Scanning mode: MRM
Capillary voltage: 3500V (-)
Nozzle voltage: 500V (-)
Ion pairs monitored in multiple reaction monitoring mode:
and drawing a standard curve by taking the peak area of the standard substance solution characteristic ion mass chromatogram as the ordinate and the concentration as the abscissa. Substituting the chromatographic peak area of the pentachlorophenol in the sample solution to be detected into a standard curve to obtain the pentachlorophenol content in the sample solution to be detected and calculating the residual amount of sodium pentachlorophenol in the eggs.
2. The LC-MS/MS method for determining the residual amount of sodium pentachlorophenate in eggs as claimed in claim 1, wherein: the solid phase extraction column is an Agilent Bond Elut Plexa PAX solid phase extraction column.
3. The LC-MS/MS method for determining the residual amount of sodium pentachlorophenate in eggs as claimed in claim 1, wherein: the concentration of the ammonia water acetonitrile solution in the step (1) is 5%.
4. The LC-MS/MS method for determining the residual amount of sodium pentachlorophenate in eggs as claimed in claim 1, wherein: the column was an Agilent ZORBAX Eclipse Plus C18, format 2.1X 50mm,1.8 μm.
5. The LC-MS/MS method for determining the residual amount of sodium pentachlorophenate in eggs as claimed in claim 1, wherein: the phase A is 5mmol/L ammonium acetate solution.
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