CN107748212B - Method for detecting medicine in goat milk and goat milk product - Google Patents

Abstract

The invention provides a method for detecting 8 major classes of 74 forbidden drugs in goat milk and products thereof, which comprises the following steps: (1) sample pretreatment: extracting a sample to be detected by using an acidic organic solvent; purifying by SPE column, dissolving the purified solution by the initial mobile phase, detecting (2)74 forbidden drugs to be detected by UPLC-MS/MS, and detecting the pretreated sample by using ultra-high performance liquid chromatography-mass spectrometry under the instrument condition specified by the method. The invention has the advantages of large amount of detected medicines, rich varieties, short detection time, high sensitivity and accurate and stable result, simultaneously makes up the blank that no various and most medicines are simultaneously detected in the goat milk and the dairy products, and has important significance for ensuring the health of consumers and improving the export trade competitiveness of the goat milk and the dairy products.

Description

Method for detecting medicine in goat milk and goat milk product

Technical Field

The invention relates to a method for detecting forbidden drugs, in particular to a method for detecting 8 major classes of 74 forbidden drugs in goat milk and products thereof.

Background

Milk and milk products are taken as indispensable parts in the dietary composition of consumers, the nutritional composition and quality safety of the milk and the milk products are concerned more and more, wherein the milk source is gradually expanded from cow milk to goat milk and the goat milk products, and compared with cow milk, the goat milk has the characteristics of small fat particles and protein particles, abundant mineral substances and vitamins and easier absorption by human bodies; the goat milk is closer to human milk in the nutrition ratio from the basic structure, is particularly suitable for infants, has less content of variant proteins such as alpha S-casein and the like, and can greatly reduce the occurrence of anaphylactic reaction. With the increase of substance level, goat milk and milk products with higher nutritional value are more and more pursued.

With the large-scale development of the breeding industry, in order to treat and prevent animal diseases, shorten the production period and promote the growth of animals, the use of veterinary drugs is very common, the current veterinary drugs commonly used in the livestock breeding industry mainly comprise sulfonamides, quinolones, macrolides, benzimidazoles, sedatives, antiviral drugs, pleuromutilins and other drugs, but due to the lack of scientific breeding knowledge and the trend of economic benefits, the overdose use phenomenon is particularly prominent, and then illegal veterinary drug production enterprises pursue curative effects and use a plurality of veterinary drugs in a compound way, and unreasonable drug holiday period limits, so that a plurality of veterinary drugs are simultaneously remained in the bodies of animals; meanwhile, in order to promote the growth and weight gain of animals, the phenomenon that forbidden drugs are added in a way of getting private interests is caused, so that the problem that the forbidden drug residues in animal-derived products exceed standards is caused frequently, in recent years, the safety events of milk and milk products are continuous, the potential threat to the health of consumers is caused, and the influence on infants and weak people is more serious.

The harm of the residual prohibited drugs in the animal derived food to the human health mainly comprises the following aspects: (1) acute and chronic toxic reaction, when the human body intakes animal-derived food with the residual medicine exceeding the standard for a long time, the acute and chronic toxic reaction can be generated when the medicine accumulation reaches a certain amount, for example, the hematopoietic function of the human body can be damaged by sulfonamides; macrolides can cause acute hepatotoxicity; the clenbuterol drugs can cause vasodilatation, blood pressure reduction, heartbeat acceleration and respiratory exacerbation so as to induce or aggravate the condition of heart patients. (2) Teratogenesis, carcinogenesis and mutagenicity, and researches show that if residual medicine food with the 'three-cause' effect is eaten for a long time, teratogenesis, carcinogenesis and mutagenicity of a human body can be finally caused, for example, the benzimidazole medicine has the teratogenesis effect; sulfadimidine and other sulfonamides can induce rodent thyroid hyperplasia and have tumor-causing tendency; individual varieties of quinolone drugs have been found to have mutagenic effects. (3) Drug-resistant bacteria are generated, sensitive strains in a human body are selectively inhibited under the long-term forbidden drug residue exposure condition, so that drug-resistant strains are propagated in large quantities, in addition, drug-resistant R plasmids are transversely transferred among strains, so that a plurality of bacteria develop from single drug resistance to multiple drug resistance, the generation of the drug-resistant bacteria causes the curative effect of some common drugs to be reduced or even lost, and the risk of poor effect or no drug availability is caused in the process of treating human diseases. In addition, the forbidden drug residues in animal derived food can cause anaphylactic reaction, such as penicillin, tetracycline, sulfanilamide, etc., which can cause anaphylactic reaction and even shock of part of people.

Therefore, many countries and organizations evaluate the safety risk of animal-derived food and make countermeasures by monitoring the forbidden drug residues, the commonly used monitoring methods mainly include a screening method and a confirmation analysis method, the screening method mainly takes ELISA rapid detection as a main part, the confirmation method mainly takes liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and gas chromatography-mass spectrometry as a main part, but with the development of the food quality safety risk situation, the existing detection method is difficult to meet the simultaneous detection requirements of multiple forbidden drugs in the same sample, and the main problems are as follows: (1) most detection methods and standards mainly detect single type of drugs, and when multiple types of drugs are detected, samples need to be processed for multiple times, so that the manpower, material resources and financial resources are greatly wasted, and the coverage of the detection method and the standard detection matrix is not comprehensive, for example, the detection matrix of animal products is generally meat and liver samples, and less kidney samples exist; most of milk and milk product substrates are mainly cow milk, and the problems of low sensitivity, poor reproducibility, complex pretreatment, large solvent consumption and the like of instruments and methods of goat milk and milk product samples (3) are not seen. Therefore, in order to meet the quality safety supervision of sample milk and dairy products, the establishment of a confirmation method for simultaneously detecting more types and more quantities of veterinary drugs is particularly urgent.

At present, the methods for detecting the forbidden drug residues in animal-derived products mainly include an ELISA method, a liquid chromatography (HPLC), a liquid chromatography-mass spectrometry (LC-MS/MS), a Gas Chromatography (GC), a gas chromatography-mass spectrometry (GC-MS/MS), a biosensor and a chip technology.

The ELISA method is a rapid screening and analyzing method established on the basis of antigen-antibody specific binding reaction, has the advantages of high sensitivity, strong specificity, large analysis flux, rapidness, safety, reliability and the like, and is a main means applied to field detection at present due to simple operation and short analysis time. However, the simultaneous ELISA method cannot detect multiple drugs simultaneously due to its strong specificity; the principle is antigen-antibody specific reaction, so that the requirement on the applicability of the method to a solvent is high, certain false positive and false negative problems are easily caused by matrix reasons, and in addition, the ELISA method has the defects of low sensitivity, poor stability and the like, and only can perform semi-quantitative analysis on an analyte.

Biosensors and chip technology integrate biologically active substances on the surface of a transducer and convert signals generated by a specific reaction between an analyte and the active substances into identifiable signals, thereby enabling the determination of the content or concentration of the analyte. The technology has the advantages of simple operation, strong specificity and high sensitivity, greatly improves the detection flux, and has small sensor volume, convenient carrying and convenient on-site real-time detection. Therefore, in recent years, the application of sensor technology to analysis and detection of forbidden drug residues is an important research field of food safety; however, as the biosensor and chip technology are emerging as detection technologies, the processing requirements for detection substrates and the existing integration technology are high due to the characteristic of maintaining the integration of bioactive substances, so that the instability of results is easily caused, and the problems of cost, service life and the like exist.

The method combines the good stability of the chromatographic technique and the higher sensitivity of the mass spectrum technique, fully exerts the characteristics of high accuracy, good stability, high sensitivity and good selectivity of the instrumental analysis method, has obvious advantages in the qualitative and quantitative analysis of forbidden drugs in animal-derived products, can still obtain accurate test results even under the complex condition of matrix interfering substances, and is a main evidence analysis means at present; however, the existing method has the defects of single type of detected medicines, small quantity of medicines and the like, and easily causes the omission of the dangerous medicines; in addition, the detection matrix is not rich enough, for example, the method and standard research are rarely focused on the forbidden drug residue condition in the goat milk and dairy products, so that the blind area and the dead angle of the food safety supervision link are caused, and the improvement of the whole level of food safety and the improvement of the export trade competitiveness in China are not facilitated.

The prior art CN103760269 discloses a method for detecting veterinary drug residues, wherein a method for detecting 46 drugs is disclosed, and the types of the detected drugs in the detection of the method are very limited, and only include tetracycline drugs, beta receptor agonist drugs, quinolone drugs and sulfonamide drugs, but cannot detect other drugs. In actual production, more types, especially more types of residual drugs need to be measured, and therefore, development of a method for simultaneously detecting more types of drugs becomes a problem to be solved in the art.

Disclosure of Invention

Aiming at the problems of small quantity and few types of forbidden drugs detected by the prior art, the invention establishes an ultra-performance liquid chromatography-mass spectrometry combined usage method for 8 types of 74 veterinary drugs including beta-receptor agonists (27 types), sulfonamides (15 types), quinolones (20 types), macrolides (3 types), benzimidazoles (3 types), sedatives (2 types), antivirals (2 types) and pleuromutilins (2 types); in addition, the established method solves the problem that the existing detection method and standard are not comprehensive aiming at the samples such as the goat milk, products thereof and the like.

Specifically, the invention is realized by the following technical scheme:

a method for detecting forbidden or/and limited drugs in goat milk and products thereof, comprising the following steps:

(1) and (3) processing of a sample:

adding an acidic organic solvent into a sample to be detected for extraction, performing vortex oscillation extraction, centrifuging to obtain a supernatant, performing vortex oscillation extraction on the precipitate obtained by centrifuging once again, combining the supernatants, purifying by an SPE column, drying the purified solution by nitrogen, dissolving by using an initial mobile phase, and determining by using UPLC-MS/MS;

(2) determination of forbidden or/and limited use drugs:

carrying out UPLC-MS/MS measurement on the sample obtained in the step (1) under the following measurement conditions:

chromatographic conditions are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 column; the mobile phase is methanol and formic acid water solution, and the elution mode is gradient elution;

mass spectrum conditions: electrospray ion source (ESI +); the capillary voltage is 3.0-3.5 kV; the source temperature is 145 DEG C-155 ℃; the desolventizing temperature is 500-600 ℃; the desolventizing gas flow velocity is 850 L.h^-1-950L·h^-1(ii) a The air velocity of the taper hole is 45 L.h^-1-55L·h^-1(ii) a Collision airflow rate: 0.13 mL/min^-1-0.15mL·min^-1。

Wherein the forbidden drugs include beta-receptor agonists, sulfonamides, quinolones, macrolides, benzimidazoles, sedatives, antiviral and pleuromutilin class 8 drugs.

Wherein the forbidden drug is metaproterenol, cimaterol, terbutaline, salbutamol, zilpaterol, sibutrol, pirbuterol, procaterol, reproterol, fenoterol, oxybenzyloxymatrine, clenbuterol, cloranolol, clenbuterol, ractopamine, mabuterol, metoprolol, marmoterol, bambuterol, odaterol, penbuterol, phenylethanolamine A, sulfacetamide, sulfamethoxazole, sulfadiazine, sulfasazole, sulfasalazole, sulfadiazine, sulfamethoxazole, sulfadoxine, sulfamoyl, sulfamethoxazole, sulfaquinoxaline, pipecolic acid, ofloxacin, gatifloxacin, salfloxacin, salsalafloxacin, salmeterol, salbutamol, one, several or all of orbifloxacin, trovafloxacin, gatifloxacin, norfloxacin, ciprofloxacin, pefloxacin, lomefloxacin, danofloxacin, enrofloxacin, gemifloxacin, moxifloxacin, sparfloxacin, difloxacin, nadifloxacin, nalidixic acid, flumequine, amantadine, rimantadine, thiabendazole, albendazole sulfone, oxfendazole, clindamycin, roxithromycin, tilmicosin, valnemulin, tiamulin, chlorpromazine and azapirone.

Wherein, the gradient elution procedure in the step (2) is as follows:

0-0.5 min: the volume ratio of the methanol is 3.0 percent and is kept unchanged;

0.5-2.0 min: the volume ratio of the methanol is 3.0-25.0%;

2.0-3.5 min: the volume ratio of the methanol is 25.0 percent and is kept unchanged;

3.5-3.6 min: the volume ratio of the methanol is 25.0-40.0%;

3.6-4.0 min: the volume ratio of the methanol is 40.0 percent and is kept unchanged;

4.0-5.5 min: the volume ratio of the methanol is 40.0-90.0%;

5.5-8.0 min: the volume ratio of the methanol is 90.0 percent and is kept unchanged;

8.0-8.1 min: the volume ratio of the methanol is 90.0-3.0%;

8.1-10.5 min: the methanol volume ratio of 3.0% was kept constant.

Wherein the temperature for blowing the supernatant liquid by nitrogen in the step (1) is 35-45 ℃, and is preferably 40 ℃.

Wherein the rotation speed of the centrifugation in the step (1) is 9000 r-min^-1-10000r·min^-1The centrifugation time is 3min-7min, and the preferred centrifugation rotating speed is 9500 r.min^-1The centrifugation time was 5 min.

Wherein the volume concentration of the extractant in the step (1) is 0.2-1.0% of acetonitrile formic acid solution; preferably, the formic acid volume concentration is 0.5%.

Wherein, in the SPE solid phase extraction column purification process in the step (1), the liquid flow rate is 0.5 drops per second-3 drops per second, and preferably the liquid flow rate is 1 drop per second.

Wherein the volume concentration of the formic acid aqueous solution in the step (2) is 0.05-0.2%, and the preferred volume concentration is 0.1%.

Wherein, the chromatographic conditions in the step (2): the chromatographic column is a Waters Acquity UPLC BEH C18 column; the mobile phase is methanol and formic acid water solution, and the elution mode is gradient elution; the flow rate is 0.3mL min < -1 >; the column temperature was 35 ℃;

mass spectrum conditions: electrospray ion source (ESI +); the capillary voltage is 3.0 KV; the extraction taper hole voltage is 3.0V; the RF lens voltage is 0.5V; the source temperature is 150 ℃; the desolventizing temperature is 600 ℃; the desolventizing gas flow velocity is 900 L.h^-1(ii) a The air velocity of the taper hole is 50 L.h^-1(ii) a The collision gas velocity was 0.14mL · min^-1(ii) a And (3) sample collection mode: and a multi-channel time-interval acquisition mode is adopted.

More specifically, the invention is realized by the following technical scheme:

a method for detecting forbidden or/and limited drugs in goat milk and products thereof is characterized by comprising the following steps:

(1) sample pretreatment:

pretreating a goat milk sample to be detected (goat milk powder is dissolved into liquid milk in advance and is treated according to a goat milk pretreatment method), adding an acidic organic solvent for extraction, performing vortex extraction, centrifuging to obtain a supernatant, performing centrifugal precipitation for one time, combining the supernatants, purifying by an SPE column, drying by purified liquid nitrogen, dissolving by using an initial mobile phase, and then determining;

(2)74 to be tested forbidden drug assays:

carrying out UPLC-MS/MS measurement on the sample obtained in the step (1) under the following measurement conditions:

chromatographic conditions are as follows: the chromatographic column is Waters Acquity UPLC BEH C₁₈A (100 mm. times.2.1 mm, 1.7 μm) column; the mobile phase is methanol and formic acid water solution, and the elution mode is gradient elution;

mass spectrum conditions: electrospray ion source (ESI +); the capillary voltage is 3.0-3.5 kV; the source temperature is 145-155 ℃; the desolventizing temperature is 500-600 ℃; the desolventizing gas flow velocity is 850 L.h^-1-950L·h^-1(ii) a The air velocity of the taper hole is 45 L.h^-1-55L·h^-1(ii) a Collision airflow rate: 0.13 mL/min^-1-0.15mL·min^-1。

Wherein the forbidden drugs comprise 8 classes of drugs such as beta-receptor agonist, sulfanilamide, quinolone, macrolide, benzimidazole, sedative, antiviral and pleuromutilin.

Wherein the forbidden drug is metaproterenol, cimaterol, terbutaline, salbutamol, zilpaterol, sibutrol, pirbuterol, procaterol, reproterol, fenoterol, oxybenzyloxymatrine, clenbuterol, cloranolol, clenbuterol, ractopamine, mabuterol, metoprolol, marmoterol, bambuterol, odaterol, penbuterol, phenylethanolamine A, sulfacetamide, sulfamethoxazole, sulfadiazine, sulfasazole, sulfasalazole, sulfadiazine, sulfamethoxazole, sulfadoxine, sulfamoyl, sulfamethoxazole, sulfaquinoxaline, pipecolic acid, ofloxacin, gatifloxacin, salfloxacin, salsalafloxacin, salmeterol, salbutamol, one, several or all of orbifloxacin, trovafloxacin, gatifloxacin, norfloxacin, ciprofloxacin, pefloxacin, lomefloxacin, danofloxacin, enrofloxacin, gemifloxacin, moxifloxacin, sparfloxacin, difloxacin, nadifloxacin, nalidixic acid, flumequine, amantadine, rimantadine, thiabendazole, albendazole sulfone, oxfendazole, clindamycin, roxithromycin, tilmicosin, valnemulin, tiamulin, chlorpromazine and azapirone.

Wherein, the gradient elution procedure in the step (2) is as follows:

0-0.5 min: the volume ratio of the methanol is 3.0 percent and is kept unchanged;

0.5-2.0 min: the volume ratio of the methanol is 3.0-25.0%;

2.0-3.5 min: the volume ratio of the methanol is 25.0 percent and is kept unchanged;

3.5-3.6 min: the volume ratio of the methanol is 25.0-40.0%;

3.6-4.0 min: the volume ratio of the methanol is 40.0 percent and is kept unchanged;

4.0-5.5 min: the volume ratio of the methanol is 40.0-90.0%;

5.5-8.0 min: the volume ratio of the methanol is 90.0 percent and is kept unchanged;

8.0-8.1 min: the volume ratio of the methanol is 90.0-3.0%;

8.1-10.5 min: the methanol volume ratio of 3.0% was kept constant.

Wherein the temperature for blowing the supernatant liquid by nitrogen in the step (1) is 35-45 ℃, and is preferably 40 ℃.

Wherein the rotation speed of the centrifugation in the step (1) is 9000 r-min^-1-10000r·min^-1The centrifugation time is 3min-7min, and the preferred centrifugation rotating speed is 9500 r.min^-1The centrifugation time was 5 min.

Wherein the volume concentration of the extractant in the step (1) is 0.2-1.0% of acetonitrile formic acid solution; preferably, the formic acid volume concentration is 0.5%.

Wherein, in the SPE solid phase extraction column purification process in the step (1), the liquid flow rate is 0.5 drops per second-3 drops per second, and preferably the liquid flow rate is 1 drop per second

Wherein the volume concentration of the formic acid aqueous solution in the step (2) is 0.05-0.2%, and the preferred volume concentration is 0.1%.

Wherein, the chromatographic conditions in the step (2): the chromatographic column is Waters Acquity UPLC BEH C₁₈A (100 mm. times.2.1 mm, 1.7 μm) column; the mobile phase is methanol and formic acid water solution, and the elution mode is gradient elution; the flow rate was 0.3 mL/min^-1(ii) a The column temperature was 35 ℃.

Mass spectrum conditions: electrospray ion source (ESI +); the capillary voltage is 3.0 KV; the extraction taper hole voltage is 3.0V; the RF lens voltage is 0.5V; the source temperature is 150 ℃; the desolventizing temperature is 600 ℃; the desolventizing gas flow velocity is 900 L.h^-1(ii) a The air velocity of the taper hole is 50 L.h^-1(ii) a The collision gas velocity was 0.14mL · min^-1。

The invention has the following beneficial technical effects:

the detection method established on the basis of high stability of the UPLC, short detection time and high sensitivity of MS/MS gives full play to the technical advantages of the UPLC and the MS, wherein the UPLC is a novel liquid phase system developed on the basis of a traditional HPLC system.

Compared to conventional High Performance Liquid Chromatography (HPLC), UPLC has the following advantages: firstly, the separation degree is high, the particle size of a chromatographic column carried by a UPLC system can reach 1.7 mu m, and the system has 70 percent of higher efficiency than a traditional HPLC (high performance liquid chromatography) chromatographic column system with the particle size of 5 mu m and 40 percent of higher efficiency than 3.5 mu m; the second is high speed, small particle size can provide higher analysis speed without affecting the separation degree, and UPLC can provide more peak capacity in the same time. Under the same condition, the separation speed of the 1.7 mu m UPLC system is improved by 9 times compared with that of a 5 mu m HPLC system; and thirdly, the sensitivity is high, the UPLC can improve the column, the peak width is reduced, the sensitivity is improved, and the sensitivity of the UPLC system with the particle size of 1.7 μm is respectively improved by 70 percent and 40 percent compared with the sensitivity of the UPLC system with the particle size of 5 μm and 3.5 μm while the column efficiency is improved. The combination of the UPLC and the mass spectrum coupling technology reflects the complementation of the advantages of the chromatogram and the mass spectrum, combines the high separation capability of the chromatogram to a complex sample with the advantages of high selectivity, high sensitivity and capability of providing relative molecular mass and structural information of MS, and provides possibility for simultaneously accurately, qualitatively and quantitatively analyzing various drugs in a complex matrix.

The SPE solid phase extraction technology is adopted, and the difference of the adsorption capacity of the analyte in different media is utilized for purification, so that the analyte is effectively separated from interference components, the detection capacity of the analyte, particularly trace analyte, is greatly enhanced, the recovery rate of a detected sample is improved, the matrix background is reduced, and the detection sensitivity is improved.

The experiment adopts many Channels minute period collection mode (multichannel data acquisition analytic system), simultaneously because the medicine kind is more, the time of appearance is comparatively concentrated in the phenomenon in 2 ~ 6min, the experiment carries out the time segmentation to different Channels, guarantee that each medicine has sufficient scanning frequency, the experiment still optimizes between 0.005 ~ 0.05ms to each medicine dwell time, can both reach 10-12 data acquisition points on the assurance all kinds of spectral peak, guarantee that each medicine can both obtain higher sensitivity and accurate ration.

The invention establishes an ultra-high performance liquid chromatography-mass spectrometry combined method which is suitable for 8 types of 74 forbidden drugs such as beta-receptor agonists (27 types), sulfonamides (15 types), quinolones (20 types), macrolides (3 types), benzimidazoles (3 types), sedatives (2 types), antivirals (2 types) and pleuromutilins (2 types) in goat milk and milk products. Compared with the existing method, the method has the advantages of large quantity of detected medicines, complete variety, short detection time, high sensitivity and accurate and stable result, makes up for the blank that no various and large quantity of medicines are simultaneously detected in the goat milk and the dairy products, and has important significance for ensuring the health of consumers and improving the export trade competitiveness of the goat milk and the dairy products.

The detection method of the invention improves the pretreatment method, so that more drugs can be extracted for further detection, meanwhile, a plurality of methods which are beneficial to the detection of a plurality of drugs are adopted in the liquid phase separation process, and a multi-channel time-sharing acquisition mode is adopted in the result acquisition, thereby being beneficial to the accumulation of signals.

Drawings

FIG. 1: a quantitative ion channel chromatogram map of a mixed standard solution of 27 beta-receptor agonists;

FIG. 2: a quantitative ion channel chromatogram of a mixed standard solution of 15 sulfonamides;

FIG. 3: quantitative ion channel chromatogram of 20 quinolone drug mixed standard solutions;

FIG. 4: quantitative ion channel chromatograms of mixed standard solutions of five classes of forbidden drugs (3 macrolides, 3 benzimidazoles, 2 antivirals, 2 sedatives, 2 pleuromutilins);

FIG. 5 shows the degree of inhibition of different drug matrices;

the above figures are the original maps of the results of the UPLC-MS/MS assay, with the following translations in english:

channels: acquisition channel of object to be detected in mass spectrometry method

Orciprenaline: oxacinalin, cimaterol: cimaterol, terbutaline: terbutaline, salbutamol: salbutamol, zilpaterol: zilpaterol, cimbuterol: sibutrol, pyrbuterol: pirbuterol, ritodrine: hydroxybenzyl-ephedrine, procaterol: procaterol, fenoterol: fenoterol, reproterol: reproterol, hydroxymethyllenbuterol: clenbuterol, clenbuterol: krusel, clenpropenol: kreppirone, clorprenaline: chlorpropaline, clenbutenol: clenbuterol, ractopamine: ractopamine, tulobuterol: tulobuterol, metoprolol: metoprolol, mabuterol: mabuterol, mapentenol: martiprotero, formoterol: formoterol, brombuterol: bromobuterol, bambusterol: bambuterol, olocataterol: oxdaterol, penbutolol: spouted britelo, phenyl EA a: phenylethanolamine a.

Sulfadiazine: sulfadiazine, sulfathiazole: sulfamethoxazole, sulfacetamide: sulphamoyl, sulphapyrine: sulfapyridine, sulfamerazine: sulfamethazine, sulfisoxazole: sulfisoxazole, sulfomoxol: sulfamethoxazole, sulfamethazine: sulfadimidine, sulfamethoxypyrazine: sulfamethoxypyridazine, sulfadoxine: sulfadoxine, sulfabenzamide: sulfonamide, sulfquinoxaline: sulfaquinoxaline, sulfamethoxazole: sulfamethoxazole, sulfamethoxine: sulfadimethoxine, sulfamomeotoxine: sulfamonomethoxine.

Pipemidic acid: pipemidic acid, ofloxacin: ofloxacin, Gatifloxacin: gatifloxacin, Sarafloxacin: sarafloxacin, Orbifloxacin/; orbifloxacin, Trovafloxacin: trovafloxacin, Garenoxacin: gawarifloxacin, norfloxacin: norfloxacin, Ciprofloxacin: ciprofloxacin, pefloxacin: pefloxacin, lomefloxacin: lomefloxacin, Danofloxacin: danofloxacin, Enrofloxacin: enrofloxacin, Gemifloxacin: gemifloxacin, Moxifloxacin: moxifloxacin, sparfloxacin spara: sparfloxacin, difloxacin: difloxacin, Nadifloxacin: nadifloxacin, nalidixic acid: nalidixic acid, Flumequine: flumequine.

Thiabendazole: thiabendazole, Albendazole sulfone: albendazole sulfone, Oxfendazole: oxfendazole.

Clindamycin: clindamycin, roxithromycin: roxithromycin, Tilmicosin: tilmicosin.

Taimulin: tiamulin, valnemulin: valnemulin.

Amantadine: amantadine, rimantadine: rimantadine.

Chlortramazine: chlorpromazine, azaperone: azapirone.

Detailed Description

The invention provides a method for detecting forbidden drugs, which comprises the following steps:

1) sample pretreatment:

pretreating a goat milk sample to be detected (goat milk powder is dissolved into liquid milk in advance and is treated according to a goat milk pretreatment method), adding an acidic organic solvent for extraction, performing vortex extraction, centrifuging to obtain a supernatant, performing centrifugal precipitation for one time, combining the supernatants, purifying by an SPE column, drying by purified liquid nitrogen, dissolving by using an initial mobile phase, and determining by UPLC-MS/MS;

the detection sample is goat milk, and when the sample is a milk powder sample, the liquid milk is quantitatively dissolved in advance.

The extraction solvent is a formic acid acetonitrile solution with the volume concentration of 0.2-1.0%; preferably, the formic acid volume concentration is 0.5%. The choice of extraction solvent is based on a combination of better protein removal and increased drug solubility.

The repeated extraction twice and the centrifugation twice are based on the consideration of fully extracting the analyte and fully removing the sediment, thereby better reducing the noise interference and improving the sensitivity of the analyte.

Preferably, the temperature of the nitrogen blow-drying of the supernatant is 35 ℃ to 45 ℃, preferably at 40 ℃.

Preferably, the rotating speed of the centrifugation is 9000 r-min^-1-10000r·min^-1The centrifugation time is 3min-7min, and the preferred centrifugation rotating speed is 9500 r.min^-1The centrifugation time was 5 min.

Preferably, the liquid flow rate during SPE solid phase extraction column purification is 0.5-3 drops per second, preferably the liquid flow rate is 1 drop per second.

In the pre-treatment process, vortex oscillation extraction has the advantages that matrix dispersion is more sufficient compared with ultrasonic extraction, and goat milk matrix can be in more comprehensive contact with an extraction solvent in the vortex process, so that the extraction of target drugs is more sufficient.

Wherein the used extractant is an acidic organic solvent, in particular a formic acid acetonitrile solution with the volume concentration of 0.2-1.0%; preferably the formic acid volume concentration is 0.5%, the use of acidic organic solvents: based on the combined consideration of better protein removal and increased drug solubility; the formic acid acidified acetonitrile is selected to replace trifluoroacetic acid acetonitrile solution in the experiment, and the method has the advantages that the trifluoroacetic acid has larger irritation and stronger toxicity, has the function of teratogenicity and mutagenicity, has larger harm to operators in the operation process, and has certain damage to mass spectrometry instruments by residual trifluoroacetic acid in the extraction process; and formic acid is used as the acidifier, so that the acidifier is low in toxicity and relatively safe for operators, and can improve the sensitivity of the object to be detected and improve the peak pattern of the object to be detected to a certain extent.

Compared with the filtration only through a microporous filter membrane, the SPE column purification can remove impurities in an extraction solution more thoroughly, particularly has an obvious effect of removing phospholipid impurities commonly existing in animal tissues, particularly can remarkably reduce the background interference of a sample by using a pretreatment method of the SPE column purification in the process of simultaneously detecting multiple drugs, and improves the sensitivity of the drug to be detected and the accuracy of a detection result.

At the same time, the initial mobile phase is used for dissolution and then is measured;

the difference of the parameters of the plurality of pre-treatments finally detects the drug which can not be detected in the prior art, thereby realizing the purpose of simultaneously detecting more drugs of more types by the method of the invention.

In conclusion, compared with the prior art, the method has the advantages that the method can simultaneously detect more types of medicines, the coverage of the medicines of the same type is wider, the pretreatment process is safer for operators, and the result is more accurate and reliable; fills the gap that the method for detecting the medicines without various and large quantities of residues in the goat milk and the dairy products.

The invention also provides a technical scheme relative to the technical problems which are not solved by the prior art, which is to improve the pretreatment method in consideration of more types and quantities of the detected medicines so as to extract and detect various different types of medicines.

The property of each kind of medicine is different in the pretreatment process, and the specific detection method is not suitable for simultaneous detection of a plurality of medicines, so that a general extraction solvent is generally adopted, and the pretreatment process is simultaneously pursued to be simple on the premise of ensuring accurate result and less background interference, which is not achieved in the prior art and is expected to be solved.

(2)74 to be tested forbidden drug assays:

carrying out UPLC-MS/MS measurement on the sample obtained in the step (1) under the following measurement conditions:

chromatographic conditions are as follows: the chromatographic column is Waters Acquity UPLC BEH C₁₈A (100 mm. times.2.1 mm, 1.7 μm) column; the mobile phase is methanol and formic acid water solution, and the elution mode is gradient elution;

preferably, the gradient elution procedure is:

0-0.5 min: the volume ratio of the methanol is 3.0 percent and is kept unchanged;

0.5-2.0 min: the volume ratio of the methanol is 3.0-25.0%;

2.0-3.5 min: the volume ratio of the methanol is 25.0 percent and is kept unchanged;

3.5-3.6 min: the volume ratio of the methanol is 25.0-40.0%;

3.6-4.0 min: the volume ratio of the methanol is 40.0 percent and is kept unchanged;

4.0-5.5 min: the volume ratio of the methanol is 40.0-90.0%;

5.5-8.0 min: the volume ratio of the methanol is 90.0 percent and is kept unchanged;

8.0-8.1 min: the volume ratio of the methanol is 90.0-3.0%;

8.1-10.5 min: the methanol volume ratio of 3.0% was kept constant.

The mobile phase proportion and the elution procedure of the gradient elution can ensure the good separation effect of various forbidden drugs.

The gradient elution adopted by the method has shorter elution time and more solvent transformation gradients, so that the separation effect is better, the mobile phase proportion and the elution procedure of the gradient elution can ensure the good separation effect of various forbidden drugs, the gradient change is quicker and the change is finer compared with the gradient change in the prior art, and the method is mainly based on that the method has more drugs, the polarity difference of the drugs is larger, and the gradient changes from a high water phase ratio to a high organic phase ratio to a high water phase ratio in a full range, so that various drugs with different polarities can be better eluted in a chromatographic column; the rapid switching of the elution gradient is mainly to ensure that the drugs with similar polarity can be better separated and quantified, and finally ensure the accuracy and reliability of the detection result.

Mass spectrum conditions: electrospray ion source (ESI +); the capillary voltage is 3.0 kV; the source temperature is 150 ℃; the desolventizing temperature is 600 ℃; the desolventizing gas flow velocity is 900 L.h^-1(ii) a The air velocity of the taper hole is 50 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1。

The invention adopts mass spectrum condition, compared with the prior art, the source temperature is 105-115 ℃; the desolventizing temperature is 340-360 ℃; the desolventizing gas flow velocity is 600 L.h^-1-700L·h^-1The mass spectrum condition has higher detection sensitivity (about 100 times) and better reproducibility.

Preferably, the method for detecting the 8 major classes of 74 forbidden drugs comprises 8 classes of drugs such as beta-receptor agonists, sulfonamides, quinolones, macrolides, benzimidazoles, sedatives, antivirals, pleuromutilins and the like.

The macrolide drugs have large molecular weight, extremely complex structure and high polarity, and generally need derivatization treatment in liquid phase analysis, while the LC-MS technology is adopted in the invention, and the macrolide drugs do not need derivatization. Therefore, in the detection process, the macrolide antibiotics and other medicines are often difficult to simultaneously detect, and in the actual production, the use and the residue of the macrolide antibiotics are common, so that the simultaneous detection of the macrolide antibiotics and other medicines by the same analysis system becomes a difficult problem in the field of pesticide residue detection.

Preferably, the medication for inhibition is metaproterenol, cimaterol, terbutaline, salbutamol, zilpaterol, sibutrol, pirbuterol, procaterol, reproterol, fenoterol, oxybenzyloxymetamine, clenbuterol, cloranolol, chlorpromaline, clenbuterol, tulobuterol, ractopamine, mabuterol, metoprolol, maprotil, bromobuterol, formoterol, bambuterol, odaterol, penbuterol, phenylethanolamine A, sulfacetamide, sulfamethoxazole, sulfadiazine, sulfasazole, sulfapyridine, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, pipecolic acid, ofloxacin, gatifloxacin, salmeterol, sulfamethoxazole, salpingine, salpigeonic acid, salfloxacin, gatifloxacin, saltifloxacin, salbutamol, salmeterol, salbutamol, salbuta, One, several or all of sarafloxacin, orbifloxacin, trovafloxacin, gatifloxacin, norfloxacin, ciprofloxacin, pefloxacin, lomefloxacin, danofloxacin, enrofloxacin, gemifloxacin, moxifloxacin, sparfloxacin, difloxacin, nadifloxacin, nalidixic acid, flumequine, amantadine, rimantadine, thiabendazole, albendazole sulfone, oxfendazole, clindamycin, roxithromycin, tilmicosin, valnemulin, tiamulin, chlorpromazine and azapirone.

The 8 major 74 drugs cover wide drug varieties, basically cover common drugs in the livestock breeding process, and have great significance for preventing the risk of illegal drug residues in animal products, especially goat milk and dairy products.

The multi-channel acquisition mode is mainly provided aiming at solving the problem that the number of detected medicines is large in the same method, and the number of the detected medicines in the same channel is limited by the conditions of an instrument, the time-sharing acquisition mode mainly solves the problem that the number of the acquired medicines to be detected in the whole time period signal acquisition process is insufficient, so that the sensitivity and the accuracy are not enough, the time-sharing acquisition mode ensures that the medicines in the time period can ensure more sufficient scanning number, and the detection sensitivity is greatly improved while the accuracy is ensured. Therefore, the method adopts a multi-channel time-sharing mode to acquire the data of the drug to be detected.

In a preferred embodiment, the method for detecting veterinary drug residues of the invention comprises the following steps:

(1) sample pretreatment: extracting a goat milk sample: accurately weighing 2g (accurate to 0.01g) goat milk in a 50mL centrifuge tube, adding 10mL 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Extracting a milk powder sample: accurately weighing 5g (accurate to 0.01g) milk powder sample, adding appropriate amount of water to dissolve, dissolving in 20mL, mixing, accurately weighing 2mL goat milk in 50mL centrifuge tube, adding 8mL 0.5% acetonitrile formate, vortex mixing for 5min,9500r·min^-1centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

(2) Determination of 74 drugs for forbidden drugs

Liquid phase conditions: a chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

Examples

Example 1

1 instruments and materials

1.1 instruments

Ultra high performance liquid chromatography (H-Class UPLC, Waters corporation); triple quadrupole mass spectrometer (xevo TQ-XS, Waters corporation); MassLynx v 4.1 data processing software (Waters Corp.); centrifuge type 3K15 (Sigma company); Milli-Q water purifier (Millipore corporation); nitrogen blowing apparatus (organization).

1.2 materials

Organic solvents such as methanol, acetonitrile, formic acid, etc. are all chromatographically pure (Fisher company); SPE cartridges (60mg, 3cc, waters corporation); a chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm, waters Co.).

74 kinds of 8 veterinary drugs such as beta-receptor agonist, sulfanilamide, quinolone, macrolide, benzimidazole, sedative, antiviral and pleuromutilin are purchased from Dr.Ehrenstorfer and carbofuran of Germany, and the purity of the veterinary drugs is over 97 percent.

Goat milk and milk powder samples purchased from E-commerce supermarket, negative samples confirmed to be free of any one of the 74 drugs by LC-MS/MS analysis are selected as matrix addition samples

The sample is goat milk: accurately weighing 2g (accurate to 0.01g) of blank goat milk in a 50mL centrifuge tube, quantitatively adding the 74 drugs for limitation, adding 10mL of 0.5% acetonitrile formate, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

The sample is goat milk powder: accurately weighing 5g (accurate to 0.01g) milk powder sample, adding appropriate amount of water to dissolve, dissolving in 20mL, mixing, accurately weighing 2mL goat milk in 50mL centrifuge tube, adding 10mL 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

2 methods and results

2.1 method

2.1.1 chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5minKeeping 25% A unchanged, and linearly changing 25% A to 40% in 3.5-3.6 min; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

2.1.2 Mass Spectrometry conditions:

ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

2.2 Mass Spectrometry Condition optimization

8 types of medicine structures such as beta-receptor agonist, sulfanilamide, quinolone, macrolide, benzimidazole, sedative, antiviral and pleuromutilin and the like all contain amino structures, so that data are collected in a positive ion mode, primary mass spectrum analysis is carried out in an MS Scan mode to obtain an [ M + H ] + peak, respective focusing voltage is optimized to enable the excimer peak to have the best response, and then secondary mass spectrum scanning is carried out on respective [ M + H ] + peaks in a Dahter Scan mode to obtain rich fragment ion information. Respective excimer ions and two pairs of daughter ions with higher intensity are selected in the experiment to meet the requirement of qualitative and quantitative analysis method of not less than 4 points in European Union 2002/957/EC resolution, and the collision voltage is optimized to ensure that the response intensity is optimal.

Limited by instrument conditions, a single channel cannot scan all 74 medicaments at one time, so that a multi-Channels mode is adopted in an experiment, the peak-off time is concentrated within 2-6 min due to the fact that the types of the medicaments are more, the experiment carries out time segmentation on different Channels, it is guaranteed that each medicament has sufficient scanning frequency, the experiment also optimizes the residence time of each medicament within 0.005-0.05 ms, it is guaranteed that 10-12 data acquisition points can be reached on each spectrum peak, it is guaranteed that each medicament can obtain high sensitivity and accurate quantification, and the optimized mass spectrum parameters and retention time of 8 types of 74 medicaments are shown in table 1.

Table 1.8 qualitative/quantitative ion (×) pairs of a large class of 74 drugs with restricted activity and their corresponding mass spectral parameters and retention times

2.3 matrix effect investigation:

when the animal derived matrix is subjected to quantitative analysis by mass spectrometry, matrix effects of different degrees and sources exist, and a matrix effect control experiment is generally designed for evaluating the influence of the matrix effects. Experimental results show that 8 types of medicines have matrix effects in different degrees in goat milk matrix, wherein beta-receptor agonist ME is 0.56-1.08, sulfonamide ME is 0.39-0.93, quinolone ME is 0.57-1.06, macrolide, benzimidazole, sedative, antiviral and pleuromutilin are 0.32-1.06, and therefore a matrix labeling solution is adopted in the detection process to eliminate the influence of the matrix effects. The degree of inhibition of the different drug matrices is shown in figure 5.

2.4 method validation

2.4.1 specificity experiments

The blank goat milk sample is added with 8 kinds of 74 forbidden drugs such as beta-receptor agonists (27 kinds), sulfonamides (15 kinds), quinolones (20 kinds), macrolides (3 kinds), benzimidazoles (3 kinds), sedatives (2 kinds), antivirals (2 kinds) and pleuromutilins (2 kinds), and compared with the blank sample chromatogram, the result shows that the 74 drugs can be well separated within 10.5 minutes, the blank sample chromatogram has no interference, and the 74 forbidden drugs are shown in figures 1-4.

2.4.2 Linear relationship and detection Limit

The experiment adopts a blank goat milk sample which is pretreated and is 1.0-80.0 mu g.L^-1Adding 74 standard solutions within the concentration range to prepare 1.0, 5.0, 10.0, 20.0 and 80.0 mu g.L^-1Adding standard solution into the substrate, sequentially injecting samples, and taking the chromatographic peak area as a vertical coordinate and the compound concentration as a horizontal coordinate to make a standard curve; diluting the processed blank goat milk sample with the standard solution step by step, and determining the quantitative Limit (LOQ) of the method according to the 10-time signal-to-noise ratio.

TABLE 6 Linear range, correlation coefficient and quantitative limit of 8 major classes of 74 drugs in goat milk

The experimental result shows that the content of 3 drugs such as beta-receptor agonist, sulfanilamide, quinolone and the like is 1.0-40.0 mu g.L^-1Has good linearity within the range, the correlation coefficient is between 0.991 and 0.999, and the 5 types of medicines such as macrolide, benzimidazole, sedative, antiviral, pleuromutilin and the like are 1.0 to 80.0 mu g.L^-1The linearity is good within the range, the correlation coefficient is between 0.992 and 0.998, the quantitative limit of 74 medicaments is less than 1.0 mu g/kg^-1See table 2 for details.

2.4.3 recovery and precision tests

Taking blank goat milk sample, adding into the sample at concentrations of 1.0, 10.0, and 20.0 μ g/kg^-1Processing samples according to the sample pretreatment method of 1.2, determining 3 batches in each concentration batch for 6 times in parallel and repeatedly measuring at different time, and performing addition recovery and fine purificationThe results of the density experiments are shown in table 3.

TABLE 3 recovery rate and variation coefficient of 8 kinds of 74 drugs added into goat milk

As can be seen from Table 3, the average recovery rate of the method is 70.1-119.7%, the intra-batch variation coefficient is 1.1-17.6%, and the inter-batch variation coefficient is 2.1-18.5%, and the accuracy and precision of the method meet the technical requirements of goat milk and dairy product species detection.

Example 2

20.0 μ L of the 27 beta-agonist drug mixtures (500 ng/ml) were added to 2g of blank negative goat milk samples. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; 8.0-8.1min, the 90% A is linearly changed to 3%, 8.1-10.5min, and the 3% A is keptAnd is not changed.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained spectrogram through measurement has the same form as the chromatogram in figure 1, which shows that the sample contains 27 beta-receptor agonist drugs, the content of each substance is calculated to be between 3.67ng/g and 5.23ng/g, the recovery rate is between 73.4 and 104.6, and the accuracy is high.

Example 3

2g of blank negative goat milk sample, 20.0. mu.L of the mixture of 15 sulfonamides (500 ng/ml concentration) was added. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lensVoltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained chromatogram has the same form as the chromatogram in figure 2, which shows that the sample contains 15 sulfonamides, the content of each substance is calculated to be between 3.55ng/g and 4.38ng/g, the recovery rate is between 71.0 percent and 87.6 percent, and the accuracy is higher.

Example 4

2g of blank negative goat milk sample, 20.0. mu.L of the 20 quinolone drug mixtures (500 ng/ml) were added. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision of vehiclesAirflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The measured spectrogram is the same as the chromatogram of fig. 3 in shape, which indicates that the sample contains 20 quinolone drugs, the content of each substance is calculated to be 3.72ng/g-5.08ng/g, the recovery rate is 74.4% -101.6%, and the accuracy is high.

Example 5

2g of blank negative goat milk sample, 20.0. mu.L of the mixture of 3 macrolides of clindamycin, roxithromycin and tilmicosin (concentration 500ng/ml) was added. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained spectrogram has the same form as the corresponding chromatogram in figure 4, which shows that the sample contains 3 macrolide drugs of clindamycin, roxithromycin and tilmicosin, the content of each substance is respectively 3.86ng/g, 3.69ng/g and 5.34ng/g by calculation, the recovery rate is between 73.8 and 106.8 percent, and the accuracy is higher.

Example 6

2g of blank negative goat milk sample, 20.0. mu.L of the mixture of 3 benzimidazole drugs of thiabendazole, albendazole sulfone and oxfendazole (concentration 500ng/ml) was added. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained spectrogram is the same as the corresponding chromatogram in the figure 4 in shape, which shows that the sample contains 3 benzimidazole medicaments of thiabendazole, albendazole sulfone and oxfendazole, the content of each substance is respectively calculated to be 3.87ng/g, 4.89ng/g and 4.05ng/g, the recovery rate is between 77.4% and 97.8%, and the accuracy is higher.

Example 7

2g of blank negative goat milk sample, 20.0. mu.L of the 2 antiviral compounds amantadine and rimantadine (500 ng/ml) were added. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained chromatogram has the same shape as the corresponding chromatogram in FIG. 4, which shows that the sample contains 2 antiviral drugs amantadine and rimantadine, and the contents of the substances are calculated to be 4.43ng/g and 4.12ng/g respectively, and the recovery rates are 88.6% and 82.4% respectively, thus indicating high accuracy.

Example 8

2g of blank negative goat milk sample, 20.0. mu.L of the mixture of chlorpromazine and azaperone 2 sedatives (500 ng/ml) was added. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained chromatogram has the same shape as the corresponding chromatogram in figure 4, which shows that the sample contains 2 sedative drugs such as chlorpromazine and azaperone, the content of each substance is calculated to be 3.72ng/g and 4.29ng/g, the recovery rates are 74.4% and 85.8%, respectively, and the accuracy is high.

Example 9

20.0 μ L of the mixture of tiamulin and 2 kinds of pleuromutilins (concentration 500ng/ml) was added to 2g of blank negative goat milk sample. Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained spectrogram through measurement has the same form as the corresponding chromatogram in figure 4, which shows that the sample contains tiamulin and 2 kinds of pleuromutilin drugs of valnemulin, the content of each substance is calculated to be 3.67ng/g and 4.15ng/g respectively, the recovery rates are 73.4 percent and 83.0 percent respectively, and the accuracy is high.

Example 10

2g of blank negative goat milk sample was added 20.0. mu.L of the mixture of the 8 major classes of 74 drugs (concentration 500 ng/ml). Adding 10mL of 0.5% formic acid acetonitrile, vortex mixing for 5min, 9500 r.min^-1Centrifuging for 5min, collecting supernatant, extracting in a 50mL centrifuge tube, mixing extractive solutions, and purifying with a purifying column.

Purifying: transferring the whole extract to SPE column activated with 85% acetonitrile water, maintaining the flow rate at 1 drop per second, collecting the whole filtrate, blowing nitrogen at 40 deg.C to near dryness, diluting to 1mL with initial mobile phase, 9500 r.min^-1Centrifuging for 5min, filtering the upper liquid layer with 0.22 μm filter membrane, and standing for determination.

Chromatographic conditions

A chromatographic column: acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); flow rate: 0.3 mL/min^-1(ii) a Column temperature: 35 ℃; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid aqueous solution, the time is 0-0.5min, the time when the 3% A is kept unchanged is 0.5-2.0min, and the time when the 3% A is linearly changed to 25%; 2.0-3.5min, keeping 25% A unchanged, and 3.5-3.6min, linearly changing 25% A to 40%; 3.6-4.0min, keeping 40% A unchanged; 4.0-5.5min, the 40% A is linearly changed to 90%; keeping 90% A unchanged for 5.5-8.0 min; and 8.0-8.1min, linearly changing the 90% A to 3%, and keeping the 3% A unchanged for 8.1-10.5 min.

Mass spectrum conditions: ionization mode: ESI +; capillary voltage: 3.0 kV; extraction taper hole voltage: 3.0V; RF lens voltage: 0.5V; source temperature: 150 ℃; desolventizing temperature: 600 ℃; taper hole air flow rate: 50 L.h^-1(ii) a Desolventizing air flow rate: 900 L.h^-1(ii) a Collision airflow rate: 0.14 mL/min^-1(ii) a An acquisition mode: multiple channelsmulti-session MRM acquisitions.

The obtained chromatogram has the same shape as the corresponding chromatogram in fig. 1-4, which shows that the sample contains the above 74 drugs, and the content of each substance is calculated to be between 3.52ng/g and 5.68ng/g, and the recovery rate is between 70.4% and 113.6%, thus indicating high accuracy.

Claims (11)

1. A method for detecting forbidden and limited drugs in goat milk and products thereof is characterized by comprising the following steps:

(1) and (3) processing of a sample:

adding an acidic organic solvent into a sample to be detected for extraction, performing vortex oscillation extraction, centrifuging to obtain a supernatant, performing repeated vortex oscillation extraction on the precipitate obtained by centrifuging, combining the supernatants, purifying by an SPE column, drying the purified liquid by nitrogen, dissolving by using an initial mobile phase, and determining by using UPLC-MS/MS;

(2) determination of drugs of forbidden and limited use:

carrying out UPLC-MS/MS measurement on the sample obtained in the step (1) under the following measurement conditions:

chromatographic conditions are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 column; the mobile phase is methanol and formic acid aqueous solution, the elution mode is gradient elution, and the gradient elution procedure is as follows:

0-0.5 min: the volume ratio of the methanol is 3.0 percent and is kept unchanged;

0.5-2.0 min: the volume ratio of the methanol is 3.0-25.0%;

2.0-3.5 min: the volume ratio of the methanol is 25.0 percent and is kept unchanged;

3.5-3.6 min: the volume ratio of the methanol is 25.0-40.0%;

3.6-4.0 min: the volume ratio of the methanol is 40.0 percent and is kept unchanged;

4.0-5.5 min: the volume ratio of the methanol is 40.0-90.0%;

5.5-8.0 min: the volume ratio of the methanol is 90.0 percent and is kept unchanged;

8.0-8.1 min: the volume ratio of the methanol is 90.0-3.0%;

8.1-10.5 min: the volume ratio of the methanol is 3.0 percent and is kept unchanged;

mass spectrum conditions: electrospray ion source ESI +;

and (3) sample collection mode: a multi-channel time-interval acquisition mode is adopted;

wherein the forbidden and limiting drugs are metaproterenol, cimaterol, terbutaline, salbutamol, zilpaterol, sibutrol, pirbuterol, procaterol, reproterol, fenoterol, oxybenzyloxymetamine, clenbuterol, cloranolol, chlorpromaline, clenbuterol, tulobuterol, ractopamine, mabuterol, metoprolol, maprotil, bromobreuterol, formoterol, bambuterol, odaterol, penbuterol, phenylethanolamine A, sulfacetamide, sulfamethoxazole, sulfadiazine, sulfasazole, sulfadiazine, sulfamethoxazole, sulfamethoxypyridazine, sulfadoxine, sulfamoyl, sulfamethoxazole, sulfaquinoxaline, pipecolic acid, ofloxacin, gatifloxacin, salmeterol, salmete, All of sarafloxacin, orbifloxacin, trovafloxacin, gatifloxacin, norfloxacin, ciprofloxacin, pefloxacin, lomefloxacin, danofloxacin, enrofloxacin, gemifloxacin, moxifloxacin, sparfloxacin, difloxacin, nadifloxacin, nalidixic acid, flumequine, amantadine, rimantadine, thiabendazole, albendazole sulfone, oxfendazole, clindamycin, roxithromycin, tilmicosin, valnemulin, tiamulin, chlorpromazine, azapirone;

wherein the acidic organic solvent in the step (1) is a formic acid acetonitrile solution with the volume concentration of 0.2-1.0%.

2. The detection method according to claim 1, wherein the temperature for blowing the nitrogen gas in the step (1) is 35 ℃ to 45 ℃.

3. The detection method according to claim 2, wherein the temperature for blowing the nitrogen gas in the step (1) is 40 ℃.

4. The detection method according to claim 3, wherein the rotation speed of the centrifugation in the step (1) is 9000 r-min^-1-10000 r·min^-1The centrifugation time is 3min-7 min.

5. The detection method according to claim 4, wherein the centrifugal rotation speed in the step (1) is 9500 r-min^-1The centrifugation time was 5 min.

6. The detection method according to any one of claims 1 to 5, wherein in the SPE column purification of step (1), the liquid flow rate is 0.5-3 drops per second.

7. The detection method according to claim 6, wherein in the SPE column purification of step (1), the liquid flow rate is 1 drop per second.

8. The detection method according to any one of claims 1 to 5, wherein the concentration by volume of the aqueous formic acid solution of step (2) is 0.05% to 0.2%, and the concentration by volume of the formic acid of step (1) is 0.5%.

9. The detection method according to claim 8, wherein the concentration by volume of the aqueous formic acid solution of step (2) is 0.1%.

10. The detection method according to any one of claims 1 to 5, wherein the chromatographic conditions of step (2): the chromatographic column is Waters Acquity UPLC BEH C₁₈A column; the mobile phase is methanol and formic acid water solution, and the elution mode is gradient elution; the flow rate was 0.3 mL/min^-1(ii) a The column temperature was 35 ℃;

mass spectrum conditions: electrospray ion source ESI +; a sample collection mode; the capillary voltage is 3.0-3.5 kV; the source temperature is 145-155 ℃; the desolventizing temperature is 500-600 ℃; the desolventizing gas flow velocity is 850 L.h^-1-950L·h^-1(ii) a The air velocity of the taper hole is 45 L.h^-1-55L·h^-1(ii) a Collision airflow rate: 0.13 mL.min^-1-0.15 mL•min^-1；

And (3) sample collection mode: and a multi-channel time-interval acquisition mode is adopted.

11. The detection method of claim 10, wherein the mass spectrometry condition is electrospray ionization ESI +; the capillary voltage is 3.0 KV; the extraction taper hole voltage is 3.0V; the RF lens voltage is 0.5V; the source temperature is 150 ℃; the desolventizing temperature is 600 ℃; the desolventizing gas flow velocity is 900 L.h^-1(ii) a The air velocity of the taper hole is 50 L.h^-1(ii) a The collision gas velocity was 0.14mL · min^-1。

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