阅读说明：本技术 一种检测动物源性食品中兽药残留方法及其应用 (Method for detecting veterinary drug residues in animal-derived food and application thereof ) 是由 王守伟 赵文涛 郭文萍 李莹莹 李石磊 郭超 李志刚 姜锐 王娟强 王妍 张颖颖 于 2019-11-13 设计创作，主要内容包括：本发明涉及一种检测动物源性食品中兽药残留的方法及其应用,所述方法包括将动物源性食品经提取、净化后进行高效液相色谱-高分辨质谱分析；所述高效液相色谱条件为：色谱柱：Agilent Eclipse plus C18；流动相：A：0.1％甲酸-水溶液B：乙腈；洗脱程序：0～0.5min A的含量保持95～98％,0.5～20min A的含量线性变化至0,20～25min A的含量维持0,25～28min A的含量维持95～98％。本发明的检测动物源性食品中兽药残留的方法可以提取并检测各类动物源食品中155种兽药残留,通过高效液相色谱-高分辨质谱联用技术可以对兽药残留同时进行定性和定量分析。(The invention relates to a method for detecting veterinary drug residues in animal-derived food and application thereof, wherein the method comprises the steps of extracting and purifying the animal-derived food, and then carrying out high performance liquid chromatography-high resolution mass spectrometry; the high performance liquid chromatography conditions are as follows: a chromatographic column: agilent Eclipse plus C18; mobile phase: a: 0.1% formic acid-water solution B: acetonitrile; elution procedure: the content of 0-0.5 min A is kept at 95-98%, the content of 0.5-20 min A is linearly changed to 0, the content of 20-25 min A is kept at 0, and the content of 25-28 min A is kept at 95-98%. The method for detecting the veterinary drug residues in the animal-derived food can extract and detect 155 veterinary drug residues in various animal-derived foods, and can perform qualitative and quantitative analysis on the veterinary drug residues simultaneously by using a high performance liquid chromatography-high resolution mass spectrometry combined technology.)

1. A method for detecting veterinary drug residues in animal derived food is characterized by comprising the following steps:

extracting and purifying animal-derived food, and performing high performance liquid chromatography-high resolution mass spectrometry; the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: agilent Eclipse plus C18; mobile phase: a: 0.1% formic acid-water solution B: acetonitrile; elution procedure:

the content of A is kept 95-98% in 0-0.5 min,

the content of A is linearly changed to 0 in 0.5-20 min,

the content of A is maintained at 0 for 20-25 min,

the content of A is maintained at 95-98% in 25-28 min.

2. The method of claim 1, wherein the conditions of the high performance liquid chromatography are:

a chromatographic column: agilent Eclipse plus C18; mobile phase: a: 0.1% formic acid-water solution B: acetonitrile; elution procedure:

the content of A is kept to be 98 percent in 0-0.5 min,

the content of A is linearly changed from 98 percent to 0 in 0.5-20 min,

the content of A is maintained at 0 for 20-25 min,

the content of A is maintained at 98% in 25-28 min.

3. The method according to claim 1 or 2, wherein the specific steps of extracting and purifying the food of animal origin are as follows:

adding the animal-derived food into the extracting solution, uniformly mixing and centrifuging, and then taking supernate to pass through a Prime HLB solid-phase extraction column; wherein the extracting solution comprises the following components in percentage by mass: 0.15-0.25% formic acid, 75-85% acetonitrile.

4. The method according to any one of claims 1 to 3, wherein after said performing high performance liquid chromatography-high resolution mass spectrometry, the following steps are performed:

(1) comparing the obtained nucleus ratio and retention time of the mother ions and the secondary fragment ions with corresponding standards in matrix labeled samples of the veterinary drugs, and carrying out qualitative analysis on the veterinary drug residues in the animal-derived food;

(2) and detecting the concentration of each veterinary drug by an external standard method, and quantitatively analyzing the veterinary drug residue in the animal-derived food.

5. The method of claim 4, wherein the veterinary drug is sulfaphenazole, sulfaphenacyl, sulfapyridine, sulfacetamide, sulfamethoxypyrimidine, sulfamoxazole, sulfamethoxazole, sulfadimethozine, sulfadimetrazine, sulfamethoxazole, sulfamethazine, sulfamethoxypyridazine, sulfammetadimethoxine, sulfamonomethoxine, sulfaquinoxaline, sulfao-dimethoxypyrimidine, sulfachloropyrazine, sulfachlorpyridazine, sulfaguanidine, sulfadiazine, sulfathiazole, sulfamethylthiadiazole, orbifloxacin, danofloxacin, enrofloxacin, flumequine, fleroxacin, ciprofloxacin, lomefloxacin, nalidixic acid, norfloxacin, pefloxacin, sarafloxacin, difloxacin, sparfloxacin, enoxacin, ofloxacin, clenbuterol, ractopamine, chlorpromazine, Penbuterol, ipratropium, formoterol, fenoterol, sibutrol, bambuterol, phenylethanolamine A, thiabendazole, salbutamol, terbutaline, tulobuterol, cimaterol, florfenicol, chlortetracycline, doxycycline, tetracycline, oxytetracycline, erythromycin, kitasamycin, lincomycin, tylosin, tilmicosin, tylosin, oleandomycin, roxitraconazole, dimetridazole, hydroxymetronidazole, hydroxymethylmetronidazole, metronidazole, oxfendazole, phenylthiocarbamate, fenbendazole, ampicillin, oxacillin, cloxacillin, dicloxacillin, penicillin, cefapine, ceftiofur, cephalexin, 19-nortestosterone, megestrol acetate, testosterone, methyltestosterone, chlorpromazine, azaperone, promethazine, acepromethazine, diazepam, doramectin, ivermectin, and, Madolicin, salinomycin, monensin, quinoxaline-2-carboxylic acid, carbadox, quindox, deoxycarbaxyloxy, levamisole, carbofuran, coumaphos, fenthiosulfone, fenthiosulfoxide, malathion, phoxim, trichlorfon, trimethoprim, atropine, procaine, lidocaine, scopolamine, hyoscyamine, sulfanilamide, mabuterol, cefazolin, amantadine, rimantadine, ribavirin, oseltamivir, epimembrin, epimedamycin, demeclocycline, trimethomycin, mebenquine, clopyralid, amproline, halofuginone, narasin, albendazole-2-aminosulfone, albendazole sulfone, albendazole sulfoxide, albendazole, sulfadiazine, chloramphenicol, beclomethasone, cortisone acetate, dexamethasone, methylprednisolone, cortisone, prednisone, hydrocortisone, and the like, One or more of fludrocortisone acetate, betamethasone, diethylstilbestrol, estradiol, lasalomectin, fipronil, clazuril, nicarbazin, fipronil sulfone, fipronil sulfoxide, fluoxynil, thiamphenicol and abamectin.

6. The method of claim 4 or 5, wherein the qualitative analysis in step (1) is performed by using the following criteria:

in the analysis result, the relative deviation of the accurate mass-to-nucleus ratio of the mother ions and the secondary fragment ions of any veterinary drug is within 5ppm, and the relative deviation of the retention time is within +/-2.5%, so that the animal-derived food can be judged to contain the veterinary drug.

7. The method according to any one of claims 4 to 6, wherein the external standard method is specifically:

taking a negative sample, respectively preparing standard solutions with different concentrations of each veterinary drug, performing high performance liquid chromatography-high resolution mass spectrometry, drawing a standard working curve according to the chromatographic peak area and the corresponding concentration, and quantitatively analyzing the veterinary drug residues in the animal-derived food through the standard working curve.

8. The method of claim 3, wherein the extract is dried and then filtered after adding 1mL of the initial mobile phase before the HPLC-MS analysis.

9. The method of claim 1, wherein the parameters of the high resolution mass spectrometry are: high resolution mass spectrometry: ScanType: full MS- -dd MS2, Polarity: negative, shear gas flow rate:40L, Auxgas flow rate:15L, Spray voltage:3.5KV, Capillary temp:325 ℃, Aux gas heatteremp: 450 ℃, FullMS Resolution:60000, dd-MS2 Resolution: 15000.

10. Use of the method according to any one of claims 1 to 9 for the safety assessment of food of animal origin.

Technical Field

The invention relates to the field of food safety, in particular to a method for detecting veterinary drug residues in animal-derived food and application thereof.

Background

In recent years, the problem of food safety is becoming more and more a concern of consumers, and the food inspection mechanism is faced with a great deal of detection work. The existing veterinary drugs used in the process of raising livestock and fish have hundreds of types, the differences of chemical structures and properties are great, the difficulty of detecting the veterinary drugs by a single method is great, and the existing commonly used veterinary drug residue detection standard methods have more than 100 types. Generally, a standard test method is only suitable for analyzing single veterinary drug residues in one type of matrix or several types of matrices, and more than 100 veterinary drug residues in a plurality of matrices need to be analyzed by a plurality of methods, so that the workload is huge, and the time and the labor are consumed for routine inspection tasks. In order to solve the problem, the liquid chromatogram tandem high resolution mass spectrum is used for carrying out trace analysis and rapid quantitative detection on various veterinary drug residues in a complex sample matrix, so that the detection flux of a laboratory can be greatly improved.

At present, many rapid screening methods for animal residues are developed at home and abroad, and there are also many methods for detecting animal drug residues in a certain type of sample matrix by using liquid chromatography-tandem high-resolution mass spectrometry, but the detection range is generally narrow, only certain type of animal-derived food, such as fish matrix, is involved, and the detection of animal drug residues in animal-derived food is few in species and difficult to comprehensively and accurately detect.

Disclosure of Invention

The invention provides a method for detecting veterinary drug residues in animal-derived food and application thereof, wherein 155 veterinary drug residues in the animal-derived food can be detected by combining a prime solid-phase extraction column purified sample with a high performance liquid chromatography/high resolution mass spectrometry combined technology.

In a first aspect, the invention provides a method for detecting veterinary drug residues in animal-derived food, comprising the steps of extracting and purifying the animal-derived food, and then carrying out high performance liquid chromatography-high resolution mass spectrometry; the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: agilent Eclipse plus C18; mobile phase: a: 0.1% formic acid-water solution B: acetonitrile; elution procedure:

the content of A is kept 95-98% in 0-0.5 min,

the content of A is linearly changed to 0 in 0.5-20 min,

the content of A is maintained at 0 for 20-25 min,

the content of A is maintained at 95-98% in 25-28 min.

Further, the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: agilent Eclipse plus C18; mobile phase: a: 0.1% formic acid-water solution B: acetonitrile; elution procedure:

the content of A is kept to be 98 percent in 0-0.5 min,

the content of A is linearly changed to 0 in 0.5-20 min,

the content of A is maintained at 0 for 20-25 min,

the content of A is maintained at 98% in 25-28 min.

In the high-throughput detection process of a large batch of veterinary drugs, the problems of overlapping of chromatographic peaks of different veterinary drugs or incapability of realizing chromatographic separation of isomers easily occur, on one hand, if most compounds generate peaks at the same time, the scanning time of a mass spectrum for each compound ion is reduced, so that the sensitivity of an instrument is reduced; on the other hand, for example, in sulfonamides, there are four pairs of isomers, which would not be distinguishable under mass spectrometric conditions if these compounds were not separated under chromatographic conditions. The invention sets specific conditions of high performance liquid chromatography for 155 veterinary drugs, solves the problem, and can accurately perform qualitative and quantitative analysis on 155 veterinary drugs in the detection result.

Further, the specific steps of extracting and purifying the animal-derived food comprise:

adding the animal-derived food into the extracting solution, uniformly mixing and centrifuging, and then taking supernate to pass through a Prime HLB solid-phase extraction column; wherein the extracting solution comprises the following components in percentage by mass: 0.15-0.25% formic acid, 75-85% acetonitrile.

In the actual analysis process, due to mutual interference among the components, the detection limits of various veterinary drugs are different, multiple veterinary drugs are difficult to detect simultaneously, and false negative results are easy to occur. The invention discovers that the method adopts a Prime HLB solid-phase extraction column and is matched with a specific 80% acetonitrile aqueous solution containing 0.2% formic acid to extract residual veterinary drugs in various animal-derived foods, purifies an extracting solution during extraction, can effectively reduce the interference effect of a matrix and reduce the pollution to mass spectrum, and can extract at least 155 residual veterinary drug components.

Further, after the performing of the high performance liquid chromatography-high resolution mass spectrometry, performing the following steps:

(1) comparing the obtained nucleus ratio and retention time of the mother ions and the secondary fragment ions with corresponding standards in matrix labeled samples of the veterinary drugs, and carrying out qualitative analysis on the veterinary drug residues in the animal-derived food;

(2) and detecting the concentration of each veterinary drug by an external standard method, and quantitatively analyzing the veterinary drug residue in the animal-derived food.

Further, the veterinary drug is sulfamethoxazole, sulfamoyl, sulfapyridine, sulfacetamide, sulfamethoxydiazine, sulfamethoxazole, sulfamethazine, sulfamethoxazole, danofloxacin, enrofloxacin, flumequine, fleroxacin, ciprofloxacin, lomefloxacin, nalidixic acid, norfloxacin, pefloxacin, sarafloxacin, difloxacin, sparfloxacin, enoxacin, ofloxacin, clenbuterol, ractopamine, chlorpropalin, pentazocine, propranolol, formoterol, fenoterol, fenoxaprop-p-ethyl, fludioxol, trimethoprim, sulfamethoxazole, Sibutrol, bambuterol, phenylethanolamine A, thiabendazole, albuterol, terbutaline, tobuterol, cimaterol, florfenicol, chlortetracycline, doxycycline, tetracycline, oxytetracycline, erythromycin, kitasamycin, lincomycin, tylosin, tilmicosin, tylosin, oleandomycin, ranibidazole, dimetridazole, hydroxymetronidazole, hydroxymethylmetronidazole, metronidazole, oxfendazole, thionocarbamate, fenbendazole, ampicillin, oxacillin, cloxacillin, dicloxacillin, penicillin, cephapirin, cefquinome, ceftiofur, cephalexin, 19-nortestosterone, megestrol acetate, testosterone, methyltestosterone, chlorpromazine, azaperone, promethazine, acepromethazine, diazepam, doramectin, ivermectin, milbemycin, monensin, quinoxalin-2-carboxylic acid, Carbalkoxy, quindoxy, carbalkoxy, levamisole, carbofuran, coumaphos, fenthiosulfone, fenthion sulfoxide, malathion, phoxim, trichlorfon, trimethoprim, atropine, procaine, lidocaine, scopolamine, anisodamine, sulfanilamide, mabuterol, cefazolin, amantadine, rimantadine, ribavirin, oseltamivir, epimembrin, demethyl aureomycin, mebenylquinoline, clopidol, amproline, halofuginone, narasin-2-aminosulfone, albendazole sulfone, albendazole sulfoxide, albendazole, sulfasalazine, chloramphenicol, beclomethasone, cortisone acetate, dexamethasone, methylprednisolone, cortisone, prednisone, hydrocortisone, fludrocortisone acetate, betamethasone, stilbestrol, estradiol, One or more of hexestrol, lasalocid, fipronil, clazuril, nicarbazin, fipronil sulfone, fipronil sulfoxide, fipronil, thiamphenicol and abamectin.

Further, the criteria for qualitative analysis are:

in the analysis result, the relative deviation of the accurate mass-to-nucleus ratio of the mother ions and the secondary fragment ions of any veterinary drug is within 5ppm, and the relative deviation of the retention time is within +/-2.5%, so that the animal-derived food can be judged to contain the veterinary drug.

Further, the external standard method specifically comprises the following steps:

taking a negative sample, respectively preparing standard solutions with different concentrations of each veterinary drug, performing high performance liquid chromatography-high resolution mass spectrometry, drawing a standard working curve according to the chromatographic peak area and the corresponding concentration, and quantitatively analyzing the veterinary drug residues in the animal-derived food through the standard working curve.

Further, before the HPLC-MS analysis, the extract was dried and then filtered after adding 1mL of the initial mobile phase.

Further, the parameters of the high resolution mass spectrum are as follows:

high resolution mass spectrometry: ScanType: full MS- -dd MS2, Polarity: negative, shear gas flow rate 40L, Aux gas flow rate 15L, Spray voltage 3.5KV, Capillary temp 325 ℃, Auxgas heater temp 450 ℃, Full MS Resolution 60000, dd-MS2 Resolution 15000.

The invention further provides application of the method in safety evaluation of animal-derived food.

The invention provides a method for detecting veterinary drug residues in animal-derived food and application thereof, and the method has the following beneficial effects:

(1) 155 veterinary drug components in the animal derived food can be comprehensively detected through specific conditions of high performance liquid chromatography-high resolution mass spectrometry, and the detection rate and the detection accuracy are high.

(2) The residual veterinary drugs in the animal-derived food can be extracted by a specific solution, and can be detected by matching with the purification process of the Prime HLB solid-phase extraction column, such as 155 veterinary drug components in edible tissues of aquatic animals, poultry or wild animals, so that the detection range is wide, and the sensitivity is high.

(3) The veterinary drug residue in the animal derived food can be simultaneously accurately qualitatively and quantitatively analyzed by high performance liquid chromatography-high resolution mass spectrometry.

Drawings

Fig. 1 is a total ion flow diagram of positive mode 132 veterinary drug residue standards provided in example 1 of the present invention;

fig. 2 is a total ion flow diagram of negative model 23 veterinary drug residue standards provided in example 1 of the present invention;

FIG. 3 is a graph comparing the average recovery of 60%, 70%, 80%, 90%, 100% acetonitrile in water versus 155 veterinary drugs provided in Experimental example 2 of the present invention;

FIG. 4 is a graph showing the comparison of the average recovery rates of 80% acetonitrile in water containing 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% formic acid, respectively, for 155 veterinary drugs in accordance with Experimental example 3 of the present invention;

fig. 5 is a comparison graph of the average yield of 155 veterinary drugs after purification of the extract by the Prime HLB solid-phase extraction column, the QuEChERS method, the FaVEx solid-phase extraction purification method, and the vQSM rapid purification method provided in experimental example 4 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.