CN113009050A - Detection method and application of quinolone compounds in dairy products - Google Patents
Detection method and application of quinolone compounds in dairy products Download PDFInfo
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- G—PHYSICS
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- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Health & Medical Sciences (AREA)
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- Biochemistry (AREA)
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Abstract
The invention discloses a detection method of quinolone compounds in milk products and application thereof, comprising the following steps: treating a sample to be detected by using acidified acetonitrile, and collecting supernatant containing the quinolone compounds; extracting the supernatant by an extracting agent to obtain a solution to be tested; and analyzing the liquid to be tested by using an ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion trap mass spectrum to realize qualitative and quantitative detection of the quinolone compounds. The detection method is simple, low in cost and good in repeatability, and the detection efficiency of the quinolone compounds in the milk products and the reliability of detection results are improved.
Description
Technical Field
The invention belongs to the field of veterinary drug residue detection, and particularly relates to a detection method and application of quinolone compounds in milk products.
Background
The milk is a beverage well liked by consumers, contains rich nutrients such as protein, milk fat, vitamins and mineral substances, contains amino acids essential to human bodies, is mostly short-chain and medium-chain fatty acids, and is easy to absorb by human bodies, and the mineral substances such as potassium, phosphorus, calcium and the like are reasonably proportioned.
Quinolone compounds (Quinolones) are chemically synthesized antibacterial drugs, influence normal metabolism and multiplication of bacteria by inhibiting activity of bacterial DNA gyrase, and are widely applied to industries such as livestock breeding and the like due to wide antibacterial spectrum and strong antibacterial action. However, these drugs and their metabolites can accumulate in animals and form drug residues to varying degrees. If a dairy product (such as milk) with overproof quinolone drug residues is eaten for a long time, stress reactions such as rash, allergy and the like can be caused, the growth of normal flora in intestinal tracts of a human body can be influenced, and low-concentration residual drugs can induce pathogenic bacteria to generate drug resistance, so that the drug resistance is potentially harmful to human health, and the drug is one of antibacterial drug categories which need important attention clinically.
The restriction value of the sum of enrofloxacin and ciprofloxacin in cow/goat milk is 100 mug/kg, which provides a judgment basis for the restriction value specified in the national standard GB 31650-2019 of food safety national standard veterinary drug maximum residue restriction in food; the ministry of agriculture of the people's republic of China announces No. 2292 clearly, think that various salts, esters and various preparations of 4 kinds of crude drugs of lomefloxacin, pefloxacin, ofloxacin and norfloxacin may cause harm or potential risks to the breeding industry and human health, stop using 4 kinds of veterinary drugs of lomefloxacin, pefloxacin, ofloxacin and norfloxacin in food animals.
The main methods for determining the residue of quinolone drugs in milk products at present comprise: enzyme linked immunosorbent assay, protein chip method, liquid chromatography-mass spectrometry, chemiluminescence method, immunochromatography method, and the like. However, these methods have qualitative and quantitative defects, which may result in inaccurate measurement results of residual quinolone drugs.
Disclosure of Invention
In order to improve the technical problem, the invention provides a method for detecting quinolone compounds in milk products and application thereof.
The detection method of the quinolone compound provided by the invention comprises the following steps: treating a sample to be detected by using acidified acetonitrile, and collecting supernatant containing the quinolone compounds; extracting the supernatant by an extracting agent to obtain a solution to be tested; and analyzing the liquid to be tested by using an ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion trap mass spectrum to realize qualitative and quantitative detection of the quinolone compounds.
According to an embodiment of the invention, the sample to be tested is a dairy product, such as cow's milk, goat's milk or other milk source. For another example, the sample to be tested may be a liquid milk product, a powdered milk product, or a solid milk product (e.g., milk tablet).
According to an embodiment of the present invention, the sample to be tested contains a quinolone compound. For example, the quinolone compound may be selected from one, two or more of norfloxacin, ofloxacin, ciprofloxacin, enrofloxacin, and the like, preferably enrofloxacin and/or ciprofloxacin.
According to an embodiment of the invention, the detection method comprises the steps of:
(1) sample pretreatment: adding acidified acetonitrile into a sample to be detected to precipitate protein in the sample to be detected, and collecting supernatant containing quinolone compounds;
the acidified acetonitrile is an acetonitrile solution containing 0.1-1% formic acid;
(2) preparing a liquid to be tested: mixing the supernatant with a solid phase extracting agent, performing vortex, centrifugation and nitrogen blowing, re-dissolving, and filtering to obtain the liquid to be tested;
the extractant is a mixture of a copolymer of divinylbenzene and N-vinyl pyrrolidone and ethylenediamine-N-propyl silane;
(3) and analyzing the liquid to be tested by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion trap mass spectrum to realize qualitative and quantitative detection of the quinolone compounds.
According to the embodiment of the invention, in the step (1), a sample to be tested is mixed with acidified acetonitrile in a vortex mode, ultrasonic extraction is carried out, then centrifugation is carried out, and supernatant containing the quinolone compound is collected.
Preferably, the volume ratio of the sample to be detected to the acidified acetonitrile is 1 (1-20), such as 1 (2-10), and the exemplary ratio is 1: 4.
Preferably, the vortex mixing time is 30 to 120s, for example 60 s.
Preferably, the time of ultrasonic extraction is 2-15 min, such as 5 min.
Preferably, the conditions of the centrifugation are: centrifuging at 4000-8000 r/min for 5-10 min, such as 6000r/min for 5 min.
According to an embodiment of the invention, in step (2), the mass ratio of the copolymer of divinylbenzene and N-vinylpyrrolidone to the mass of ethylenediamine-N-propylsilane is (1-3):1, for example 1.5: 1.
Preferably, the mass to volume ratio of the extractant to the supernatant is (15-35) mg:1mL, for example 25mg:1 mL.
According to an embodiment of the invention, in the step (2), an extractant is added to the supernatant, vortex mixing is carried out, centrifugation is carried out, the supernatant is sucked and purged to be nearly dry by nitrogen, the dried substance is re-dissolved by acetonitrile aqueous solution, and filtration is carried out, so as to obtain the liquid to be tested.
Preferably, the vortex mixing time is 30 to 120s, for example 60 s.
Preferably, the conditions of the centrifugation are: centrifuging at 4000-8000 r/min for 5-10 min, such as 6000r/min for 5 min.
Preferably, the concentration of the acetonitrile aqueous solution is 5-20%, for example 10%.
Preferably, the filtration is through a 0.22 μm filter membrane.
According to an embodiment of the present invention, in the step (3), the test conditions of the ultra high performance liquid chromatography include:
C18chromatography column, preferably C18100mm multiplied by 2.1mm, the grain diameter of the filler is 1.7 mu m;
mobile phase: a-0.1% formic acid water solution, B-0.1% formic acid acetonitrile solution; gradient elution;
preferably, the gradient elution procedure is as follows: keeping A for 90% and B for 10% for 0-0.5 min; for 0.5-1.4 min, A is reduced to 5% from 90%, and B is increased to 95% from 10%; 1.4-2.2 min, keeping A at 5% and B at 95%; 2.2-2.3 min, wherein A is increased to 90% from 5%, and B is decreased to 10% from 95%; 2.3-3 min, keeping A at 90% and B at 10%;
flow rate: 0.3-0.5 mL/min, preferably 0.4 mL/min;
sample introduction amount: 3-6 muL, preferably 5 muL;
column temperature: 30-40 ℃, preferably 35 ℃.
According to an embodiment of the present invention, in step (3), the test conditions of the triple quadrupole/linear ion trap mass spectrometer are as follows:
an ion source: electrospray positive (ESI +); scanning mode: MS/MS/MS; spraying voltage: 3800-4200V, preferably 4000V; temperature: 400-600 ℃, preferably 500 ℃; collision energy: 25-35 eV, preferably 30 eV; de-clustering voltage: 60-100V, preferably 80V; excitation energy: 60-80V, preferably 70V; scanning speed: 250 to 4000Da/s, preferably 4000 Da/s.
Preferably, when the quinolone compound is enrofloxacin or ciprofloxacin, the ion information of enrofloxacin or ciprofloxacin is shown in the following table:
according to the embodiment of the present invention, before performing the hplc tandem triple quadrupole/linear ion trap mass spectrometry, the preparation of the standard solution of the quinolone compound and the drawing of the standard curve are further included, and preferably, the following steps are included:
preparing a standard solution of a quinolone compound by using a blank sample extracting solution to prepare a standard solution working solution with 5-7 concentration gradient points; and fitting a standard curve graph of the quinolone compound by taking secondary ions of the quinolone compound obtained by the triple quadrupole/linear ion trap mass spectrometry as quantitative ions, taking the concentration as a horizontal ordinate and the target peak area as a vertical ordinate.
For example, the concentration gradient of the quinolone compound is 2, 10, 20, 50, 100 ng/mL.
According to an embodiment of the present invention, the step (3) comprises: and comparing the target peak area value of the sample obtained by the high performance liquid chromatography test with a standard curve to obtain the concentration of the quinolone compound in the liquid to be tested, and further calculating the content of the quinolone compound in the sample to be tested.
In the present invention, "%" represents volume percent.
The invention also provides application of the detection method for detecting the quinolone compounds in the milk products.
The invention has the beneficial effects that:
1. the method uses the acidified acetonitrile to extract the quinolone compounds enrofloxacin and ciprofloxacin in the milk products (such as milk), and provides beneficial help for protein precipitation in the milk products and the extraction efficiency of the target compounds enrofloxacin and ciprofloxacin by utilizing a full vortex and ultrasonic treatment mode.
2. The pretreatment process of the invention uses a dispersed solid phase extraction mode to purify the sample extracting solution, the operation method is simple, the cost is low, the repeatability is good, and the detection efficiency and the reliability of the detection result are improved.
3. According to the invention, the ultra-high performance liquid chromatography is used for analyzing the enrofloxacin and the ciprofloxacin by connecting the triple quadrupole/linear ion trap mass spectrum in series, richer compound information is obtained by utilizing the scanning mode of MS/MS/MS, the respective parent ions, the respective daughter ions and the respective secondary daughter ions of the enrofloxacin and the ciprofloxacin are determined, and the qualitative score reaches 4. The method has accurate qualitative determination, and reduces the misjudgment probability of false positive (negative) samples.
4. The invention uses the secondary ion of the target compound for quantitative analysis, has stronger selectivity, reduces the interference degree of impurities in the sample and greatly enhances the signal-to-noise ratio (S/N) of the target peak.
5. The invention uses the blank matrix solution to prepare the standard curve working solution, provides a matrix environment similar to that in an actual sample, and uses the correction curve to carry out quantitative analysis on the sample, thereby reducing the interference of the instrument analysis on the target compound and improving the accuracy of the quantitative analysis result of the quinolone compound.
Drawings
FIG. 1 shows the fragmentation of enrofloxacin parent ion (m/z 360) into target daughter ion fragment (m/z 316).
FIG. 2 shows the fragmentation of enrofloxacin daughter ion fragment (m/z 316) into target secondary daughter ion fragment (m/z 296).
FIG. 3 shows the fragmentation of ciprofloxacin parent ion (m/z 332) into target daughter ion fragment (m/z 288).
FIG. 4 shows the fragmentation of ciprofloxacin daughter ion fragment (m/z 288) into the target secondary daughter ion fragment (m/z 268).
FIG. 5 is an enrofloxacin UHPLC-MS/MS/MS chromatogram with a target compound retention time of 1.73 minutes.
FIG. 6 is a UHPLC-MS/MS/MS chromatogram of ciprofloxacin, with a target compound retention time of 1.76 minutes.
FIG. 7 is a standard graph of enrofloxacin in example 1; the abscissa of the graph is ciprofloxacin concentration (ng/mL), and the ordinate is the target fragment peak area.
FIG. 8 is a standard graph of ciprofloxacin in example 1; the abscissa of the graph is ciprofloxacin concentration (ng/mL), and the ordinate is the target fragment peak area.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Enrofloxacin standards and ciprofloxacin standards were purchased from: tianjin Aza technologies, Inc.
Copolymers of divinylbenzene with N-vinyl pyrrolidone are available from: beijing Dike Macco technologies, Inc.
Example 1
1. And (3) processing of a sample:
respectively adding an enrofloxacin standard solution and a ciprofloxacin standard solution (shown in table 2, the concentrations of the ciprofloxacin standard solutions are 2.5 mug/mL, 5 mug/mL and 25 mug/mL, and the concentrations of the enrofloxacin standard solutions are 2.5 mug/mL, 5 mug/mL and 25 mug/mL. methanol is adopted to dissolve the standard solutions) into the weighed blank milk sample (not containing enrofloxacin and ciprofloxacin), and uniformly mixing to obtain the ciprofloxacin standard sample and the enrofloxacin standard sample of the embodiment. Accurately sucking 2.0mL of labeled sample, adding 8mL of acetonitrile containing 1% formic acid as extractive solution, mixing by vortex for 60s, ultrasonic extracting for 5min, centrifuging at 6000r/min for 5min, and collecting supernatant.
Purifying and concentrating supernatant: adding 150mg of divinylbenzene and N-vinyl pyrrolidone copolymer and 100mg of ethylenediamine-N-propyl silane into the collected supernatant, mixing by vortex for 60s, centrifuging at 6000r/min for 5-10 min, sucking 5.0mL of the supernatant, purging by nitrogen at 40 ℃ until the supernatant is nearly dry, fixing the volume by 10% acetonitrile water solution, fully re-dissolving, and then passing through a 0.22 mu m filter membrane to obtain the liquid to be tested. Using an ultra performance liquid chromatography to serially connect a triple quadrupole/linear ion trap mass spectrum to analyze a liquid to be tested, wherein the test conditions are as follows:
2. liquid chromatography conditions:
a chromatographic column: c18100mm multiplied by 2.1mm, the grain diameter of the filler is 1.7 mu m;
mobile phase: a-0.1% formic acid water solution, B-0.1% formic acid acetonitrile solution. Gradient elution, gradient elution procedure: keeping A for 90% and B for 10% for 0-0.5 min; for 0.5-1.4 min, A is reduced from 90% to 5%, and B is increased from 10% to 95%; 1.4-2.2 min, keeping A at 5% and B at 95%; 2.2-2.3 min, wherein A is increased to 90% from 5%, and B is decreased to 10% from 95%; 2.3-3 min, keeping A at 90% and B at 10%.
Flow rate: 0.4 mL/min;
sample introduction amount: 5 mu L of the solution;
column temperature: 35 ℃ is carried out.
3. Mass spectrum conditions:
an ion source: electrospray positive (ESI +), scan mode: MS/MS/MS, spray voltage: 4000V, temperature: 500 ℃, collision energy: 30eV, declustering voltage: 80V, excitation energy: 70V, scanning speed: 4000Da/s, compound ion information, see Table 1, FIGS. 1-4:
TABLE 1
4. Detection procedure and results
And (3) diluting the enrofloxacin and ciprofloxacin standard substance solution by using a blank matrix extracting solution (the blank matrix extracting solution refers to the supernatant obtained by extracting the blank milk sample in the step 1) to obtain standard curve series concentration gradients of 2, 10, 20, 50 and 100ng/mL, and loading on a machine. The standard curve was fitted with secondary ions 296, 268 as the quantification ions, the upper computer concentration as the abscissa, and the chromatographic peak area as the ordinate (FIGS. 7-8).
The curve equations of enrofloxacin and ciprofloxacin are respectively: y is 4109.2x +22363.5 (R0.9981), y is 4409.1x +17476.4 (R0.9995).
After the sample is processed by the machine, the corresponding content is obtained according to a linear equation, and the result is shown in the following table 2 and fig. 5-6.
TABLE 2
Example 2
1. And (3) processing of a sample:
accurately sucking 2.0mL of commercially available milk (brand, Yili; producing area, Hebei Tangshan), adding 8mL of acetonitrile containing 1% formic acid as extractive solution, vortex mixing for 60s, ultrasonic extracting for 5min, centrifuging at 6000r/min for 5min, and collecting supernatant.
Purifying and concentrating supernatant: adding 150mg of copolymer powder of divinylbenzene and N-vinyl pyrrolidone and 100mg of ethylenediamine-N-propyl silane powder into the collected supernatant, mixing for 60s by vortex, centrifuging for 5-10 min at 6000r/min, sucking 5.0mL of supernatant, purging with nitrogen at 40 ℃ until the supernatant is nearly dry, fixing the volume with 10% acetonitrile solution, fully re-dissolving, and passing through a 0.22 mu m filter membrane to obtain the solution to be tested. Using an ultra performance liquid chromatography to serially connect a triple quadrupole/linear ion trap mass spectrum to analyze a liquid to be tested, wherein the test conditions are as follows:
2. liquid chromatography conditions:
a chromatographic column: c18100mm multiplied by 2.1mm, the grain diameter of the filler is 1.7 mu m;
mobile phase: a-0.1% formic acid water solution, B-0.1% formic acid acetonitrile solution. Gradient elution, gradient elution procedure: keeping A for 90% and B for 10% for 0-0.5 min; for 0.5-1.4 min, A is reduced from 90% to 5%, and B is increased from 10% to 95%; 1.4-2.2 min, keeping A at 5% and B at 95%; 2.2-2.3 min, wherein A is increased to 90% from 5%, and B is decreased to 10% from 95%; 2.3-3 min, keeping A at 90% and B at 10%.
Flow rate: 0.4 mL/min;
sample introduction amount: 5 mu L of the solution;
column temperature: 35 ℃ is carried out.
3. Mass spectrum conditions:
an ion source: electrospray positive (ESI +), scan mode: MS/MS/MS, spray voltage: 4000V, temperature: 500 ℃, collision energy: 30eV, declustering voltage: 80V, excitation energy: 70V, scanning speed: 4000Da/s, compound ion information is shown in Table 3:
TABLE 3
4. Detection procedure and results
And (3) diluting the enrofloxacin and ciprofloxacin standard substance solution by using the blank matrix extracting solution to obtain a series of concentration gradients of standard curves of 2, 10, 20, 50 and 100ng/mL, and processing. And fitting a standard curve graph by taking secondary ionic ions 296 and 268 as quantitative ions, the upper computer concentration as a horizontal coordinate and the chromatographic peak area as a vertical coordinate. The curve equations of enrofloxacin and ciprofloxacin are respectively: y is 4109.2x +22363.5 (R0.9981), y is 4409.1x +17476.4 (R0.9995).
And (3) detection results: the sample is negative, and enrofloxacin and ciprofloxacin are not detected.
In the embodiment of the invention, the detection limit of the enrofloxacin and the ciprofloxacin is 1 ng/mL.
The invention adopts an ultra-high performance liquid chromatography to connect with a triple quadrupole/linear ion trap mass spectrometer in series to carry out qualitative and quantitative analysis on a test sample liquid, and obtains the contents of enrofloxacin and ciprofloxacin. The method can accurately and sensitively measure the contents of the enrofloxacin and the ciprofloxacin in the milk, and improves the accuracy of quantitative analysis results of the enrofloxacin and the ciprofloxacin.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting a quinolone compound, comprising the steps of: treating a sample to be detected by using acidified acetonitrile, and collecting supernatant containing the quinolone compounds; extracting the supernatant by an extracting agent to obtain a solution to be tested; and analyzing the liquid to be tested by using an ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion trap mass spectrum to realize qualitative and quantitative detection of the quinolone compounds.
2. The detection method according to claim 1, wherein the sample to be detected is a dairy product, such as cow's milk, goat's milk or other milk source. As another example, the sample to be tested may be a liquid milk product, a powdered milk product, or a solid milk product.
Preferably, the sample to be detected contains quinolone compounds; the quinolone compound is selected from one, two or more of norfloxacin, ofloxacin, ciprofloxacin and enrofloxacin, and is preferably enrofloxacin and/or ciprofloxacin.
3. The detection method according to any one of claims 1-2, comprising the steps of:
(1) sample pretreatment: adding acidified acetonitrile into a sample to be detected to precipitate protein in the sample to be detected, and collecting supernatant containing quinolone compounds;
the acidified acetonitrile is an acetonitrile solution containing 0.1-1% formic acid;
(2) preparing a liquid to be tested: mixing the supernatant with an extracting agent, carrying out re-dissolution after vortex, centrifugation and nitrogen blowing, and filtering to obtain the liquid to be tested;
the solid phase extracting agent is a mixture of a copolymer of divinylbenzene and N-vinyl pyrrolidone and ethylenediamine-N-propyl silane;
(3) and analyzing the liquid to be tested by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion trap mass spectrum to realize qualitative and quantitative detection of the quinolone compounds.
4. The detection method according to claim 3, wherein in the step (1), the sample to be detected is mixed with acidified acetonitrile by vortex, extracted by ultrasonic wave, and then centrifuged, and the supernatant containing the quinolone compound is collected.
Preferably, the volume ratio of the sample to be detected to the acidified acetonitrile is 1 (1-20), such as 1 (2-10), and the exemplary ratio is 1: 4.
Preferably, the vortex mixing time is 30 to 120s, for example 60 s.
Preferably, the time of ultrasonic extraction is 2-15 min, such as 5 min.
Preferably, the conditions of the centrifugation are: centrifuging at 4000-8000 r/min for 5-10 min, such as 6000r/min for 5 min.
5. The detection method according to claim 3 or 4, wherein in the step (2), the mass ratio of the sum of the mass of the copolymer of divinylbenzene and N-vinylpyrrolidone to the mass of ethylenediamine-N-propylsilane is (1-3: 1, for example, 1.5: 1.
Preferably, the mass to volume ratio of the extractant to the supernatant is (15-35) mg:1mL, for example 25mg:1 mL.
Preferably, in step (2), adding a solid phase extracting agent into the supernatant, vortex mixing, centrifuging, sucking the supernatant, purging the supernatant to be nearly dry with nitrogen, redissolving the dry matter with acetonitrile aqueous solution, and filtering to obtain the liquid to be tested.
Preferably, the vortex mixing time is 30 to 120s, for example 60 s.
Preferably, the conditions of the centrifugation are: centrifuging at 4000-8000 r/min for 5-10 min, such as 6000r/min for 5 min.
Preferably, the concentration of the acetonitrile aqueous solution is 5-20%, for example 10%.
Preferably, the filtration is through a 0.22 μm filter membrane.
6. The detection method according to any one of claims 3 to 5, wherein in the step (3), the test conditions of the ultra-high performance liquid chromatography include:
C18chromatography column, preferably C18100mm multiplied by 2.1mm, the grain diameter of the filler is 1.7 mu m;
mobile phase: a-0.1% formic acid water solution, B-0.1% formic acid acetonitrile solution; gradient elution;
preferably, the gradient elution procedure is as follows: keeping A for 90% and B for 10% for 0-0.5 min; for 0.5-1.4 min, A is reduced to 5% from 90%, and B is increased to 95% from 10%; 1.4-2.2 min, keeping A at 5% and B at 95%; 2.2-2.3 min, wherein A is increased to 90% from 5%, and B is decreased to 10% from 95%; 2.3-3 min, keeping A at 90% and B at 10%;
flow rate: 0.3-0.5 mL/min, preferably 0.4 mL/min;
sample introduction amount: 3-6 muL, preferably 5 muL;
column temperature: 30-40 ℃, preferably 35 ℃.
7. The detection method according to any one of claims 3 to 6, wherein in the step (3), the test conditions of the triple quadrupole/linear ion trap mass spectrometry are as follows:
an ion source: electrospray positive (ESI +); scanning mode: MS/MS/MS; spraying voltage: 3800-4200V, preferably 4000V; temperature: 400-600 ℃, preferably 500 ℃; collision energy: 25-35 eV, preferably 30 eV; de-clustering voltage: 60-100V, preferably 80V; excitation energy: 60-80V, preferably 70V; scanning speed: 250 to 4000Da/s, preferably 4000 Da/s.
8. The detection method according to any one of claims 1 to 7, further comprising the preparation of a standard solution of a quinolone compound and the drawing of a standard curve before the ultra performance liquid chromatography tandem triple quadrupole/linear ion trap mass spectrometry, preferably comprising the following steps:
preparing a standard solution of a quinolone compound by using a blank sample extracting solution to prepare a standard solution working solution with 5-7 concentration gradient points; and fitting a standard curve graph of the quinolone compound by taking secondary ions of the quinolone compound obtained by the triple quadrupole/linear ion trap mass spectrometry as quantitative ions, taking the concentration as a horizontal ordinate and the target peak area as a vertical ordinate.
Preferably, the concentration gradient of the quinolone compound in the standard solution working solution is 2, 10, 20, 50 and 100 ng/mL.
9. The detection method according to any one of claims 3 to 8, wherein the step (3) comprises: and comparing the target peak area value of the sample obtained by the high performance liquid chromatography test with a standard curve to obtain the concentration of the quinolone compound in the liquid to be tested, and further calculating the content of the quinolone compound in the sample to be tested.
10. Use of the assay according to any one of claims 1 to 9 for the detection of quinolone compounds in milk products.
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