CN116678976A - Quantitative detection method for enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof - Google Patents
Quantitative detection method for enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof Download PDFInfo
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- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical group FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 title claims abstract description 103
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
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Abstract
The invention relates to the field of veterinary drug residue detection, in particular to a quantitative detection method for enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof, which comprises the following steps: chicken tissue, pork or egg is sequentially extracted by 2% formic acid acetonitrile, saturated n-hexane of acetonitrile is degreased, oasis PRiME HLB solid phase extraction column is purified, or milk is sequentially extracted by 10% trichloroacetic acid-acetonitrile (9:1, v:v), saturated n-hexane of acetonitrile is degreased, and is purified by Strata-X solid phase extraction column, and eluent is dried and concentrated under nitrogen flow; adding methanol into the concentrated and dried sample for redissolution, adding trimethylsilyl diazomethane after redissolution, filtering after derivative reaction, and detecting filtrate by adopting GC-MS/MS. The method has the advantages of more accurate qualitative and quantitative, good accuracy and sensitivity, high recovery rate and good repeatability.
Description
Technical Field
The invention relates to the field of veterinary drug residue detection, in particular to a pre-column derivatization-gas chromatography-tandem mass spectrometry (GC-MS) method for detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof.
Background
The detection methods of enrofloxacin and ofloxacin residues at home and abroad mainly comprise a high performance liquid chromatography-ultraviolet detection method, a high performance liquid chromatography-diode array detection method, a high performance liquid chromatography-fluorescence detection method and a liquid chromatography-mass spectrometry combination method, and the methods of detecting enrofloxacin and ofloxacin residues in animal foods by using GC, GC-MS and GC-MS/MS have not been reported at home and abroad.
Disclosure of Invention
In order to solve the problems of extracting and purifying enrofloxacin and ofloxacin residues from chicken tissues (muscles, livers and kidneys), pork, eggs (whole eggs, egg white and egg yolk) and milk and products thereof and derivatizing the enrofloxacin and ofloxacin residues. In the test, liquid-liquid extraction and solid-phase extraction are adopted to extract chicken tissues (muscle, liver and kidney), pork, eggs (whole egg, egg white and yolk) and milk and products thereof, and the residues of enrofloxacin and ofloxacin are extracted and purified; methylation of enrofloxacin and ofloxacin with trimethylsilyl diazomethane (TMSD) the derivatization of enrofloxacin and ofloxacin is performed as follows:
FIG. 1 enrofloxacin methylation equation
Figure 2 ofloxacin methylation equation
In order to be able to detect residues of enrofloxacin and ofloxacin in chicken tissues (muscle, liver, kidney), pork, eggs (whole egg, egg white, yolk) and milk and products thereof by using a gas chromatography-tandem mass spectrometry, the invention provides a pre-column derivatization-gas chromatography-tandem mass spectrometry (GC-MS/MS) detection method. Through methodological parameter verification, the method can realize accurate qualitative and quantitative detection, and has the advantages of high recovery rate, high accuracy and good sensitivity, and meets the requirements of veterinary drug residue detection.
The technical scheme provided by the invention is as follows:
a quantitative detection method for enrofloxacin and ofloxacin residues in chicken tissues (muscle, liver and kidney), pork, eggs (whole egg, egg white and yolk) or milk and products thereof comprises the following steps:
sequentially extracting chicken tissue (muscle, liver, kidney), pork or egg (whole egg, egg white and yolk) with 2% formic acid acetonitrile, degreasing with acetonitrile saturated n-hexane, purifying with Oasis PRiME HLB solid phase extraction column, or sequentially extracting milk and its product with 10% trichloroacetic acid-acetonitrile (9:1, v:v), degreasing with acetonitrile saturated n-hexane, purifying with Strata-X solid phase extraction column, drying and concentrating the eluate under nitrogen flow at 40deg.C;
adding methanol to the concentrated and dried sample for redissolution, adding trimethylsilyl diazomethane (TMSD) after redissolution, filtering by using a 0.22 mu m organic phase nylon needle filter after the derivatization reaction, and detecting the filtrate by using GC-MS/MS.
Further, the chicken tissue is chicken muscle, chicken liver or chicken kidney; the egg is whole chicken egg, egg white or egg yolk; the milk is fresh milk or milk powder.
Further, the GC-MS/MS detection gas chromatography conditions were: taking TG-1MS as a capillary chromatographic column; the high-purity helium is used as carrier gas, and the flow rate of the carrier gas column is 1.0mL/min.
Further, the temperature programming step of GC-MS/MS detection is as follows: the initial temperature is 100 ℃, and the temperature is kept for 1min; raising the temperature to 220 ℃ at 30 ℃/min, and keeping for 1min; raising the temperature to 300 ℃ at 30 ℃/min and keeping the temperature for 6min.
Further, the mass spectrum conditions of the GC-MS/MS detection are as follows: an electron bombardment ion source; ionization energy of 70eV; high-purity argon is collision gas; the temperature of the ion source is 300 ℃; the temperature of the transmission line is 300 ℃; the solvent delay time was 3.0min; qualitative in full scan mode, quantitative in selective reaction monitoring mode.
Further, the steps of purification of the Oasis PRiME HLB solid phase extraction column are as follows: the HLB solid phase extraction column was rinsed with 3mL of 10% methanol solution, the total flow of the rinse was awaited, a vacuum pump was used for 5min, and 2mL of methanol was added to elute the target.
Further, the step of purifying the Strata-X solid phase extraction column is as follows: the Strata-X solid phase extraction column was equilibrated with 3mL of methanol and water, rinsed with 3mL of 10% methanol solution, and the target was eluted by waiting for the total flow of the rinse solution, pumping with vacuum for 5min, and then adding 3mL of methanol.
Further, the GC-MS/MS detection is advanced for pre-column derivatization, and the pre-column derivatization comprises the following steps: adding 100 mu L of methanol into a centrifuge tube dried by nitrogen, re-dissolving along the tube wall, stirring uniformly for 2min, adding 200 mu L of TMSD, reacting at room temperature in a dark place for 30min, fixing the volume to 1mL by using methanol, stirring uniformly for 2min, filtering by using a 0.22 mu m organic phase nylon needle filter, and detecting and analyzing the filtrate by using GC-MS/MS.
Further, the peak areas detected by GC-MS/MS are used for calculating the residual amounts of enrofloxacin and ofloxacin in chicken tissues (muscles, livers and kidneys), pork, eggs (whole eggs, egg white and egg yolk) or milk and products thereof.
Further, the detection formulas of chicken tissues (muscle, liver and kidney), pork, eggs (whole egg, egg white and yolk) and milk and products thereof are respectively as follows:
chicken tissues (muscle, liver and kidney), pork, eggs (whole egg, egg white and yolk) and milk and products thereof are extracted and purified through liquid-liquid extraction and solid phase extraction, trimethylsilyl diazomethane is derivatized, and the derived products (enrofloxacin trimethylsilicone methyl ester and ofloxacin trimethylsilicone methyl ester) are detected by GC-MS/MS. Gas chromatography conditions: TG-1MS (30.0mX0.25mX0.25mmi.d.) was used as capillary column; high purity helium (> 99.999%,60 psi) was the carrier gas with a carrier gas column flow rate of 1.0mL/min. The temperature programming step is to keep the initial temperature of 100 ℃ for 1min; raising the temperature to 220 ℃ at 30 ℃/min, and keeping for 1min; raising the temperature to 300 ℃ at 30 ℃/min and keeping the temperature for 6min. The temperature of the sample inlet is 300 ℃; a non-split sample injection mode; the split flow rate is 50.0mL/min; the non-shunt time is 1.0min; a constant current mode; the flow rate of the carrier gas is 1.0mL/min; opening the valve after 2min, wherein the carrier gas saving time is 2min, and the carrier gas saving flow is 20.0mL/min; sample injection volume: 1.0. Mu.L. Mass spectrometry conditions: an electron bombardment ion source (EI); ionization energy of 70eV; high purity argon (> 99.999%,40 psi) is the collision gas; the temperature of the ion source is 300 ℃; the temperature of the transmission line is 300 ℃; the solvent delay time was 3.0min; qualitative in the full SCAN (SCAN) mode, selective reaction monitoring (Auto SRM) mode.
A method for extracting and purifying enrofloxacin and ofloxacin residues from chicken tissues (muscle, liver and kidney), pork, eggs (whole egg, egg white and egg yolk) and milk and products thereof and derivatizing the enrofloxacin and ofloxacin residues comprises the steps of liquid-liquid extraction, solid phase extraction and purification, and pre-column derivatization by taking trimethylsilyl diazomethane as a derivatizing reagent. The extraction and purification process comprises three steps: firstly, extracting enrofloxacin and ofloxacin residues in chicken tissues, pork or eggs by using 2% acetonitrile formate, extracting enrofloxacin and ofloxacin residues in milk and milk powder by using 10% trichloroacetic acid-acetonitrile (9:1, v:v), degreasing by using acetonitrile saturated n-hexane, and thirdly, purifying the extract by using an Oasis PRiME HLB solid-phase extraction column (3 cc/60 mg), and drying and concentrating the eluent by using nitrogen. Derivatization reaction process: the concentrated sample is re-dissolved by methanol after being dried by nitrogen, 200 mu L TMSD is added after re-dissolution, methanol is used for fixing the volume to 1mL after light-shielding reaction for 30min at room temperature, vortex mixing is carried out for 2min, an organic phase nylon needle filter with the volume of 0.22 mu m is used for filtering, and the filtrate is used for GC-MS/MS detection and analysis, thus confirming that enrofloxacin and ofloxacin derivative products are enrofloxacin trimethyl silicon methyl ester and ofloxacin trimethyl silicon methyl ester respectively.
The steps of extraction and purification are: accurately weighing 2 (+ -0.02) g of homogenized sample, placing the homogenized sample into a 50mL polypropylene centrifuge tube, adding 5mL of 2% acetonitrile formate (5 mL of 10% trichloroacetic acid-acetonitrile (9:1, v: v) into milk and milk powder), mixing by vortex for 10min, performing ultrasonic vibration extraction for 10min, performing centrifugation at a speed of 10000r/min at 4 ℃ for 10min, and transferring the supernatant into a new centrifuge tube. Adding 5mL of extract into the residue, repeatedly extracting for 1 time, centrifuging at 10000r/min for 10min, and mixing the two extracts; adding 10mL of acetonitrile saturated n-hexane into the extracting solution, mixing by vortex for 10min, oscillating by ultrasonic for 10min, standing for delamination, and removing the upper n-hexane.
And (3) solid phase extraction and purification: purifying enrofloxacin and ofloxacin residues in chicken tissues, pork or eggs by using an Oasis PRiME HLB solid-phase extraction column (3 cc/60 mg), passing the liquid through the column at uniform speed, eluting by using 3mL of 10% methanol solution, waiting for the eluting solution to flow out completely, pumping for 5min by using a vacuum pump, and adding 2mL of methanol for eluting; enrofloxacin and ofloxacin residues in milk are purified by using a Strata-X solid phase extraction column (60 mg/3 mL), activated and balanced by 3mL of methanol and 3mL of ultrapure water, the liquid is passed through the column at a constant speed, eluted by 3mL of 10% methanol solution, and the eluting solution is waited to flow out completely, pumped by a vacuum pump for 5min, and then eluted by adding 3mL of methanol.
In summary, the optimal sample pretreatment conditions were selected: at a sample mass of 2.0g, 10mL of 2% acetonitrile formate or 10% trichloroacetic acid-acetonitrile (9:1, v: v) was used as the extraction reagent.
TMSD is a derivatization reagent for pre-column derivatization step: drying the eluent at 40 ℃ in a nitrogen blowing instrument, re-dissolving the eluent by using methanol, adding 200 mu L of TMSD, reacting for 30min at room temperature in a dark place, fixing the volume to 1mL by using methanol, mixing the mixture uniformly by vortex for 2min, filtering the mixture by using a 0.22 mu m organic phase nylon needle filter, and detecting and analyzing the filtrate by using GC-MS/MS.
Advantageous effects
The invention compares the effect of the combination of liquid-liquid extraction and solid phase extraction on the results. As a result, it was found that the derivative obtained by the extraction method combining liquid-liquid extraction and solid phase extraction was excellent in peak shape, less in interfering impurity peak, and high in response value of the sample derivative. Meanwhile, the influence of acetonitrile, 1% acetonitrile acetate, 2% acetonitrile formate and 80% acetonitrile solution on the extraction effect of target substances in chicken tissues, pork and eggs and the influence of 10% trichloroacetic acid-acetonitrile (9:1, v: v), 1% acetonitrile acetate and 2% acetonitrile formate on the extraction effect of target substances in milk and milk powder are compared. The result shows that when the chicken tissues, pork and eggs take 2% acetonitrile formate as an extraction reagent, the recovery rate of the extraction result is highest; milk and milk powder have the highest recovery rate of the extraction result when 10% trichloroacetic acid-acetonitrile (9:1, v:v) is used as the extraction reagent. Therefore, the experiment finally selects 2% acetonitrile formate as an extraction reagent of chicken tissues, pork and eggs, 10% trichloroacetic acid-acetonitrile (9:1, v:v) as an extraction reagent of milk, and extracts and purifies enrofloxacin and ofloxacin residues in the chicken tissues (muscles, livers and kidneys), pork, eggs (whole eggs, egg white and egg yolk) and the milk by a method of combining liquid-liquid extraction and solid phase extraction.
Because enrofloxacin and ofloxacin belong to fluoroquinolone medicines with large molecular weight, high boiling point and strong polarity, the detection by directly utilizing a gas chromatography or a gas chromatography-mass spectrometry is not feasible. Therefore, the invention uses trimethylsilyl diazomethane (TMSD) as a derivatization reagent, adopts methylation reaction to derivatize enrofloxacin and ofloxacin, and changes the chemical structure, thereby improving the thermal stability of the enrofloxacin and ofloxacin. Up to now, studies on enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs and milk and performing derivatization reaction with TMSD to generate enrofloxacin trimethylsilyl ester and ofloxacin trimethylsilyl ester have not been reported at home and abroad.
In the invention, a TG-1MS (30.0mX0.25mu m X0.25mm i.d.) capillary chromatographic column is adopted, a full Scanning (SCAN) mode is adopted for qualitative, a selective reaction monitoring (Auto SRM) mode is adopted for quantitative determination, and finally, the obtained enrofloxacin and ofloxacin derivative products have good chromatographic peak shapes (i.e. high sensitivity), moderate analyte retention time and no other impurity peak interference.
The invention provides a method for detecting enrofloxacin and ofloxacin residues by utilizing pre-column derivatization-gas chromatography-tandem mass spectrometry (GC-MS/MS). The method has high recovery rate, precision and sensitivity and good repeatability, and is suitable for application and popularization in batch sample analysis. Through methodological parameter verification, the method can realize accurate qualitative and quantitative detection, has high recovery rate and accuracy and good sensitivity, meets the requirements of enrofloxacin and ofloxacin residue detection in livestock and poultry tissues, is successfully applied to actual sample detection, and meets the technical requirements of different laboratories.
Compared with the reported analysis method, the pretreatment method for the test is simple to operate, and the test cost is saved. The method is more accurate in nature and quantification, good in accuracy and sensitivity, high in recovery rate and good in repeatability, provides a new technical support for detecting enrofloxacin and ofloxacin residues in animal-derived foods, meets the technical requirements of different laboratories, and provides a scientific basis for formulating GC-MS/MS detection standards of enrofloxacin and ofloxacin residues in animal-derived foods.
Drawings
FIG. 1 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) mixed standard (B) for blank chicken muscle sample (A) and blank chicken muscle sample;
FIG. 2 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) mixed standard (B) for blank chicken liver sample (A) and blank chicken liver sample;
FIG. 3 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) mixed standard (B) for blank chicken kidney sample (A) and blank chicken kidney sample;
FIG. 4 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatograms (MC) of a mixed standard (B) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) added to a blank pork sample (A) and a blank pork sample;
FIG. 5 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (50.0 μg/kg) and ofloxacin (5.0 μg/kg) mixed standard (B) for blank chicken whole egg sample (A) and blank chicken whole egg sample;
FIG. 6 Total ion flow chromatogram (TIC) and qualitative amounts of ions (MC) of enrofloxacin (50.0 μg/kg) and ofloxacin (5.0 μg/kg) (B) added to blank egg white sample (A) and blank egg white sample;
FIG. 7 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatograms (MC) of enrofloxacin (50.0 μg/kg) and ofloxacin (5.0 μg/kg) (B) added to blank egg yolk sample (A) and blank egg yolk sample;
FIG. 8 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) (B) added to blank milk sample (A) and blank milk sample;
FIG. 9 Total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) (B) added to blank milk powder sample (A) and blank milk powder sample.
FIG. 10 is a graph of a blank chicken muscle matrix matching enrofloxacin and ofloxacin standards;
FIG. 11 is a graph of blank chicken liver matrix matching enrofloxacin and ofloxacin standards;
FIG. 12 is a graph of a blank chicken kidney matrix matching enrofloxacin and ofloxacin standards;
FIG. 13 is a graph of a blank pork matrix matching enrofloxacin and ofloxacin standards;
FIG. 14 is a blank chicken whole egg matrix matching enrofloxacin and ofloxacin calibration curve;
FIG. 15 is a blank egg white matrix matching enrofloxacin and ofloxacin calibration curves;
FIG. 16 is a blank yolk matrix matching enrofloxacin and ofloxacin calibration curves;
FIG. 17 is a graph of a blank raw milk matrix matching enrofloxacin and ofloxacin standards;
the blank milk powder base of fig. 18 matches enrofloxacin and ofloxacin standard curves.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific embodiments and with reference to the data. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the necessary list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are all the same as the first indicated.
Firstly, raising and sample collection of experimental livestock and poultry
The trial was conducted with approval by the ethical committee of the university of Yangzhou, xianglong poultry industry development limited and Changzhou, confucius agriculture and animal husbandry limited.
10 Shore chickens (Liriodendron fowl development Co., ltd.) of 100 days old and 6 ternary hybridization pigs (Du X Long X big) (He Zhou Kangle farm and animal husbandry Co., ltd.) of 6 months old are randomly selected, and the complete feed without any medicine provided by the Liriodendron Dairy feed factory in Yangzhou City is fed in the whole test period in a mode of single-cage (circle) feeding and free drinking. After 20 days of raising, slaughtering, collecting, chopping and homogenizing chest muscles, livers, kidneys and longest muscles of pig backs of each chicken, taking the chicken as a blank sample, and storing the chicken in a refrigerator at-34 ℃ for later use.
40 Shore chickens (Xianglong poultry development Co., yangzhou) with eggs at 26 weeks of age are randomly selected, and the complete feed without any medicine provided by the Xianglong poultry development Co., yangzhou is fed during the test period in a mode of single cage feeding and free drinking. After 21 days of test, eggs are collected at 18:00 a day, continuously collected for 10 days, and the whole chicken eggs, egg white and egg yolk are respectively separated and homogenized, and are packaged in a centrifuge tube and are used as blank samples to be stored in a refrigerator at the temperature of minus 34 ℃ in a sealing way for standby.
Cow milk and products (raw milk and milk powder) collected in the study are provided by YangDakangyuan milk company of Yangzhou city. All the samples are detected by the GC-MS/MS method established by the invention, so that no target compound to be detected is ensured in the samples. Split charging the fresh milk without the target compound to be detected after detection into a 50mL centrifuge tube with a plug to serve as a blank sample to be detected; and (3) sub-packaging and marking the milk powder without the target compound to be detected after detection by using a small-size self-sealing bag as a blank sample to be detected. All samples were kept in a-34℃refrigerator for further use
(II) the extraction, purification, concentration and derivatization steps of the invention
(1) Accurately weighing 2 (+ -0.02) g of a homogenized blank sample, placing the blank sample into a 50mL polypropylene centrifuge tube, adding 5mL of 2% acetonitrile formate (5 mL of 10% trichloroacetic acid-acetonitrile (9:1, v: v) into milk and milk powder), carrying out vortex mixing for 10min, carrying out ultrasonic extraction for 10min, carrying out high-speed centrifugation at a speed of 10000r/min for 10min at a temperature of 4 ℃, and transferring the supernatant into a new centrifuge tube. Adding 5mL of the extract into the residue, repeatedly extracting for 1 time, centrifuging at 10000r/min for 10min, and mixing the two extracts.
Adding 10mL of acetonitrile saturated n-hexane into the extracting solution for degreasing, placing on a vortex mixer for vortex mixing for 10min, oscillating for 10min by ultrasonic waves, standing for layering, and removing the upper n-hexane.
(2) Purifying enrofloxacin and ofloxacin residues in chicken tissues, pork or eggs by using an Oasis PRiME HLB solid-phase extraction column (3 cc/60 mg), passing the liquid through the column at uniform speed, eluting by using 3mL of 10% methanol solution, waiting for the eluting solution to flow out completely, pumping for 5min by using a vacuum pump, and adding 2mL of methanol for eluting; purifying enrofloxacin and ofloxacin residues in milk by using a Strata-X solid phase extraction column (60 mg/3 mL), activating and balancing by using 3mL of methanol and 3mL of ultrapure water, passing the liquid through the column at uniform speed, eluting by using 3mL of 10% methanol solution, waiting for the eluting solution to flow out completely, pumping for 5min by using a vacuum pump, and adding 3mL of methanol for eluting
(3) Drying the eluent at 40 ℃ in a nitrogen blowing instrument, re-dissolving the eluent by using methanol, adding 200 mu L of TMSD, reacting for 30min at room temperature in a dark place, fixing the volume to 1mL by using methanol, mixing the mixture uniformly by vortex for 2min, filtering the mixture by using a 0.22 mu m organic phase nylon needle filter, and detecting and analyzing the filtrate by using GC-MS/MS.
The invention compares the effect of the combination of liquid-liquid extraction and solid phase extraction on the results. As a result, it was found that the derivative obtained by the extraction method combining liquid-liquid extraction and solid phase extraction was excellent in peak shape, less in interfering impurity peak, and high in response value of the sample derivative. Meanwhile, the influence of acetonitrile, 1% acetonitrile acetate, 2% acetonitrile formate and 80% acetonitrile solution on the extraction effect of target substances in chicken tissues, pork and eggs is compared, and the detailed table 1 is shown; the effect of 10% trichloroacetic acid-acetonitrile (9:1, v:v), 1% acetic acid acetonitrile, 2% formic acid acetonitrile on the extraction of targets in milk and milk powder was compared and detailed in Table 2. The result shows that when the chicken tissues, pork and eggs take 2% acetonitrile formate as an extraction reagent, the recovery rate of the extraction result is highest; milk and milk powder have the highest recovery rate of the extraction result when 10% trichloroacetic acid-acetonitrile (9:1, v:v) is used as the extraction reagent. Therefore, the experiment finally selects 2% acetonitrile formate as an extraction reagent of chicken tissues, pork and eggs, 10% trichloroacetic acid-acetonitrile (9:1, v:v) as an extraction reagent of milk, and extracts and purifies enrofloxacin and ofloxacin residues in the chicken tissues (muscles, livers and kidneys), pork, eggs (whole eggs, egg white and egg yolk) and the milk by a method of combining liquid-liquid extraction and solid phase extraction.
Table 1 effect (%) of different extraction reagents on recovery of enrofloxacin and ofloxacin in chicken tissues, pork and eggs (n=3)
Table 2 effect (%) of different extraction reagents on recovery of enrofloxacin and ofloxacin in milk and milk powder (n=3)
(III) GC-MS/MS analysis conditions
1. Gas chromatography conditions
Chromatographic column: TG-1MS (30.0mX0.25mX0.25mmi.d., 100% dimethylpolysiloxane); carrier gas: high purity helium (> 99.999%,60 psi), carrier gas column flow rate: 1.0mL/min.
A temperature programming step: the initial temperature is 100 ℃, and the temperature is kept for 1min; raising the temperature to 220 ℃ at 30 ℃/min, and keeping for 1min; raising the temperature to 300 ℃ at 30 ℃/min, and keeping for 6min; details are shown in Table 3. Sample inlet temperature: 300 ℃; split mode: sample introduction without diversion; split flow rate: 50.0mL/min; non-split time: 1.0min; carrier gas mode: a constant current mode; carrier gas flow rate: 1.0mL/min; opening the valve after 2min, saving the carrier gas by 2min, and saving the carrier gas by 20.0mL/min; sample injection volume: 1.0. Mu.L.
TABLE 3 temperature programmed step
2. Mass spectrometry conditions
Ionization mode: an electron bombardment ion source (EI); electron beam energy (ionization energy): 70eV; collision gas: high purity argon (> 99.999%,40 psi); ion source temperature: 300 ℃; transmission line temperature: 300 ℃; solvent delay: 3.0min; data acquisition mode: qualitative, selective reaction monitoring (Auto SRM) mode quantification was performed in a full SCAN (SCAN) mode. The molecular weight and mass spectral parameters of enrofloxacin, ofloxacin derivatives are shown in table 4.
TABLE 4 retention times and associated mass spectral parameters for target compounds
Note that: * Quantitative ion pairs
(IV) quantitative method
1. Drawing of a Standard Curve
Respectively weighing 2 (+ -0.02) g of homogeneous blank samples accurately, preparing 9 blank extracting solutions with different matrixes according to the method (II), and placing the extracting solutions in a refrigerator at the temperature of minus 34 ℃ for standby. The standard working solution is mixed by gradually diluting enrofloxacin (8.0 mug/mL) and ofloxacin (0.8 mug/mL) from high concentration to low concentration by using a blank matrix extracting solution, and series chicken muscle matrix matching mixed standard working solution with enrofloxacin concentration of 3.2 (LOQ), 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0ng/mL and ofloxacin concentration of 0.6, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0ng/mL is prepared; the preparation method comprises the steps of preparing a series of chicken liver matrix matching mixed standard working solutions with enrofloxacin concentration of 3.8, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0ng/mL and ofloxacin concentration of 0.8, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 ng/mL; the preparation method comprises the steps of preparing a series of chicken kidney matrix matching mixed standard working solutions with enrofloxacin concentration of 3.4, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0ng/mL and ofloxacin concentration of 0.6, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 ng/mL; the mixed standard working solution is matched with series pork matrixes with enrofloxacin concentration of 3.2, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0ng/mL and ofloxacin concentration of 0.6, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 ng/mL; preparing a series of chicken whole egg matrix matching mixed standard working solutions with enrofloxacin concentration of 2.6 (LOQ), 25.0, 50.0, 100.0, 200.0, 400.0ng/mL and ofloxacin concentration of 0.6, 2.5, 5.0, 10.0, 20.0 and 40.0 ng/mL; the enrofloxacin is prepared into serial egg white matrix matching mixed standard working solutions with the concentration of 2.0, 25.0, 50.0, 100.0, 200.0, 400.0ng/mL and the concentration of ofloxacin is 0.4, 2.5, 5.0, 10.0, 20.0 and 40.0 ng/mL; the enrofloxacin is prepared into a series of egg yolk matrix matching mixed standard working solutions with the concentration of 3.0, 25.0, 50.0, 100.0, 200.0, 400.0ng/mL and the ofloxacin concentration of 0.8, 2.5, 5.0, 10.0, 20.0 and 40.0 ng/mL; the enrofloxacin concentration is 3.6, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0ng/mL and the ofloxacin concentration is 0.6, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0 ng/mL; the milk powder matrixes with enrofloxacin concentration of 4.0, 25.0, 50.0, 100.0, 200.0, 400.0, 800.0, 1600.0ng/mL and ofloxacin concentration of 0.8, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0ng/mL are prepared to match mixed standard working solutions (wherein LOQ mixed concentration points are obtained by respectively diluting a single standard working solution with blank matrix extract).
Derivatizing the prepared matrix matching mixed standard working solution according to the method (II), filtering the solution by using a 0.22 mu m organic phase nylon needle filter, and detecting the filtrate by a GC-MS/MS machine. Each concentration was set up in 6 replicates and the average calculated. The matrix matching standard curves are respectively drawn by taking the concentration of a series of matrix matching standard working solutions (enrofloxacin and ofloxacin) as an abscissa (x), and taking the chromatographic peak areas of quantitative sub-ions of enrofloxacin and ofloxacin derivative products (quantitative ion pair m/z 385.3>384.1 of enrofloxacin derivative products and quantitative ion pair m/z 387.2>386.2 of ofloxacin derivative products) as an ordinate (y), wherein the curves are used as quantitative curves of various samples to be tested.
As can be seen from Table 5 and figures 10-18, the concentration of the standard working solution of enrofloxacin matched with the matrix of the pork of the blank chicken is 3.2-1600.0 ng/mL, the concentration of the standard working solution of enrofloxacin matched with the matrix of the liver of the blank chicken is 3.8-1600.0 ng/mL, the concentration of the standard working solution of enrofloxacin matched with the matrix of the kidney of the blank chicken is 3.4-1600.0 ng/mL, the concentration of the standard working solution of enrofloxacin matched with the matrix of the whole egg of the blank chicken is 2.6-400.0 ng/mL, the concentration of the standard working solution of enrofloxacin matched with the matrix of the blank egg is 2.0-400.0 ng/mL, the concentration of the standard working solution of enrofloxacin matched with the matrix of the blank egg Huang Jizhi is 3.0-400.0 ng/mL, the concentration of the standard working solution of enrofloxacin matched with the matrix of the blank raw fresh milk is 3.6-400.0 ng/mL, the standard working solution of enrofloxacin matched with the matrix of the milk powder is 4.0-400.0 ng/mL, the concentration of standard working solution of the matrix matching ofloxacin of the blank chicken muscle, chicken kidney, pork, chicken whole egg and raw fresh milk is 0.6-160.0 ng/mL, the concentration of standard working solution of the matrix matching ofloxacin of the blank chicken liver, egg yolk and milk powder is 0.8-160.0 ng/mL, the concentration of standard working solution of the matrix matching ofloxacin of the blank egg white is 0.4-40.0 ng/mL, the mass chromatographic peak area (y) of characteristic ion (quantitative sub-ion) and the concentration (x) of the matrix matching standard working solution are respectively in good linear relation (R 2 Not less than 0.9990), and the linear regression equation, the determination coefficient and the linear range are shown in Table 5. If the concentration analyzed exceeds the line of the sampleThe concentration of the sample is required to be diluted within the range, and the concentration of the sample is obtained by multiplying the detected result by the dilution factor.
Table 5 chicken tissue, pork, egg and milk base matching linear regression equation, determination coefficients and linear range of enrofloxacin and ofloxacin standard working solutions (n=6)
2. Determination of recovery and precision
2 (+ -0.02) g of homogeneous blank chicken tissues, pork, eggs and milk are accurately weighed, a proper amount of enrofloxacin and ofloxacin standard working solution is respectively added, so that the addition concentrations of enrofloxacin and ofloxacin in each blank sample are LOQ, 0.5MRL, 1.0MRL and 2.0MRL, and 6 parallel experiments are carried out on each concentration. The sample is subjected to pretreatment and derivatization according to the step (II). The derivatized solution was filtered and the filtrate was subjected to on-machine detection by GC-MS/MS. Recording peak area, substituting the peak area into a matrix matching standard curve to calculate enrofloxacin and ofloxacin concentrations, and further calculating the addition recovery rate of the sample.
The total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the enrofloxacin (100.0 mug/kg) and ofloxacin (10.0 mug/kg) mixed standard (B) added to the blank chicken muscle sample (A) and the blank chicken muscle sample are shown in figure 1; the total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the enrofloxacin (100.0 mug/kg) and ofloxacin (10.0 mug/kg) mixed standard (B) of the blank chicken liver sample (A) and the blank chicken liver sample are shown in figure 2; the total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the enrofloxacin (100.0 mug/kg) and ofloxacin (10.0 mug/kg) mixed standard (B) of the blank chicken kidney sample (A) and the blank chicken kidney sample are shown in figure 3; the total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatogram (MC) of the enrofloxacin (100.0 mug/kg) and ofloxacin (10.0 mug/kg) mixed standard (B) added to the blank pork sample (A) and the blank pork sample are shown in figure 4; total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (50.0 μg/kg) and ofloxacin (5.0 μg/kg) mixed standard (B) added into blank chicken whole egg (A) and blank chicken whole egg sample are shown in figure 5; the total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the mixed standard (B) of enrofloxacin (50.0 mug/kg) and ofloxacin (5.0 mug/kg) added to the blank egg white sample (A) and the blank egg white sample are shown in figure 6; total ion flow chromatogram (TIC) and qualitative and quantitative ion Mass Chromatogram (MC) of enrofloxacin (50.0 μg/kg) and ofloxacin (5.0 μg/kg) mixed standard (B) added to blank egg Huang Yangpin (A) and blank egg yolk sample are shown in figure 7; the total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of the enrofloxacin (100.0 mug/kg) and ofloxacin (10.0 mug/kg) (B) added to the blank milk sample (A) and the blank milk sample are shown in figure 8; the total ion flow chromatogram (TIC) and the qualitative and quantitative ion Mass Chromatograms (MC) of enrofloxacin (100.0 μg/kg) and ofloxacin (10.0 μg/kg) (B) added to the blank milk powder sample (A) and the blank milk powder sample are shown in FIG. 9.
Intra-day (intra-batch) precision: samples of 4 additive concentrations (LOQ, 0.5MRL, 1.0MRL, 2.0 MRL) at different time points on the same day were assayed using the same instrument and the same standard curve, and each additive concentration was repeatedly tested for RSD 6 times.
Daytime (inter-batch) precision: samples of 4 additive concentrations (LOQ, 0.5MRL, 1.0MRL, 2.0 MRL) were assayed on different days of the week using the same instrument and different standard curves (standard curves were drawn every day), and RSD was repeatedly measured 6 times for each additive concentration.
Under this condition, the recovery rate of enrofloxacin and ofloxacin added in chicken tissue, pork, eggs and milk by the method of the invention is shown in tables 6-7.
Table 6 recovery and precision of enrofloxacin addition in chicken tissue, pork, eggs and milk (n=6)
Note that: alpha, maximum residual limit
Table 7 recovery and precision of ofloxacin addition in chicken tissue, pork, eggs and milk (n=6)
Note that: alpha, maximum residual limit
3. Determination of detection limit and quantitative limit
Adding a proper amount of enrofloxacin and ofloxacin standard working solution into a blank sample, extracting, purifying and derivatizing the blank sample according to the step (II), starting to detect by using an optimized instrument method, setting 6 parallel concentrations each, and observing the signal-to-noise ratio of a characteristic ion (quantitative sub-ion) chromatographic peak and the corresponding drug concentration, wherein the corresponding concentration when the S/N is more than or equal to 3 is LOD of the method; the corresponding concentration of S/N is more than or equal to 10 is LOQ of the method.
According to the above-mentioned 6 parallel blank sample addition recovery test, it was obtained that under the existing conditions, the LOD of enrofloxacin in chicken muscle, chicken liver, chicken kidney, pork, chicken whole egg, egg white, chicken yolk, raw milk and milk powder was 0.7, 1.0, 0.8, 0.5, 0.4, 0.8 and 1.1 μg/kg, respectively, and the LOQ was 1.6, 1.9, 1.7, 1.6, 1.3, 1.0, 1.5, 1.8 and 2.0 μg/kg, respectively; the LOD of ofloxacin in chicken muscle, chicken liver, chicken kidney, pork, chicken whole egg, egg white, egg yolk, raw milk and milk powder was 0.1, 0.2, 0.1, 0.2, 0.1 and 0.2. Mu.g/kg, respectively, and the LOQ was 0.3, 0.4, 0.3, 0.2, 0.4, 0.3 and 0.4. Mu.g/kg, respectively.
(V) comparison with other analytical methods
As can be seen from Table 8, the recovery and sensitivity using mass spectrometry are generally higher than those of other methods, and the LOD and LOQ values are also lower in this test result. The test fills the blank of detecting enrofloxacin and ofloxacin residues in animal-derived foods by GC-MS/MS, and meets the detection requirements of different laboratories.
TABLE 8 comparison of methods for detecting enrofloxacin and ofloxacin in foods of different animal origins
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Claims (10)
1. The quantitative detection method for enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof is characterized by comprising the following steps of:
chicken tissue, pork or egg is sequentially extracted by 2% formic acid acetonitrile, saturated n-hexane of acetonitrile is degreased, oasis PRiME HLB solid phase extraction column is purified, or milk and products thereof are sequentially extracted by 10% trichloroacetic acid-acetonitrile (9:1, v:v), saturated n-hexane of acetonitrile is degreased, strata-X solid phase extraction column is purified, and eluent is dried and concentrated under nitrogen flow; adding methanol into the concentrated and dried sample for redissolution, adding trimethylsilyl diazomethane after redissolution, filtering after derivative reaction, and detecting filtrate by adopting GC-MS/MS.
2. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissue, pork, eggs or milk and products thereof according to claim 1, wherein the chicken tissue is chicken muscle, chicken liver or chicken kidney; the egg is whole chicken egg, egg white or egg yolk; the milk and its product are fresh milk or milk powder.
3. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the gas chromatographic conditions for GC-MS/MS detection are: taking TG-1MS as a capillary chromatographic column; the high-purity helium is used as carrier gas, and the flow rate of the carrier gas column is 1.0mL/min.
4. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the step of programming the temperature for GC-MS/MS detection is as follows: the initial temperature is 100 ℃, and the temperature is kept for 1min; raising the temperature to 220 ℃ at 30 ℃/min, and keeping for 1min; raising the temperature to 300 ℃ at 30 ℃/min and keeping the temperature for 6min.
5. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the mass spectrometry conditions of the GC-MS/MS detection are as follows: an electron bombardment ion source; ionization energy of 70eV; high-purity argon is collision gas; the temperature of the ion source is 300 ℃; the temperature of the transmission line is 300 ℃; the solvent delay time was 3.0min; qualitative in full scan mode, quantitative in selective reaction monitoring mode.
6. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the steps of purification by an Oasis PRiME HLB solid phase extraction column are as follows: the HLB solid phase extraction column was rinsed with 3mL of 10% methanol solution, the total flow of the rinse was awaited, a vacuum pump was used for 5min, and 2mL of methanol was added to elute the target.
7. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the step of Strata-X solid phase extraction column purification is as follows: the Strata-X solid phase extraction column was equilibrated with 3mL of methanol and water, rinsed with 3mL of 10% methanol solution, and the target was eluted by waiting for the total flow of the rinse solution, pumping with vacuum for 5min, and then adding 3mL of methanol.
8. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the GC-MS/MS detection is carried out by pre-column derivatization, the pre-column derivatization comprising the steps of: adding 100 mu L of methanol into a centrifuge tube dried by nitrogen, re-dissolving along the tube wall, stirring uniformly for 2min, adding 200 mu L of TMSD, reacting at room temperature in a dark place for 30min, fixing the volume to 1mL by using methanol, stirring uniformly for 2min, filtering by using a 0.22 mu m organic phase nylon needle filter, and detecting and analyzing the filtrate by using GC-MS/MS.
9. The method for quantitatively detecting enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the peak areas detected by GC-MS/MS are used for calculating the enrofloxacin and ofloxacin residues in the chicken tissues, pork, eggs or milk and products thereof.
10. The quantitative detection method of enrofloxacin and ofloxacin residues in chicken tissues, pork, eggs or milk and products thereof according to claim 1, wherein the detection formulas of the chicken tissues, pork, eggs or milk and products thereof are respectively as follows:
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