Non-targeted detection method for simultaneously screening multiple pesticide and veterinary drug residues in raw milk and application of non-targeted detection method

Abstract

The invention discloses a non-targeted screening method for pesticide and veterinary drug residues in raw milk based on LC-TOFMS (liquid chromatography-time of flight mass spectrometry) and application of the non-targeted screening method, and belongs to the technical field of food safety detection. According to the method, a QuEChERS technology is adopted to carry out pretreatment on a raw milk sample, a liquid chromatography-time-of-flight mass spectrometry technology is utilized to carry out detection, and a self-built mass spectrometry database is combined to realize non-targeted qualitative screening. A self-established mass spectrum database comprises precise mass numbers, retention time, parent ion loading modes and secondary mass spectrum fragment information of 46 kinds of pesticides and 54 kinds of veterinary drugs. The method is simple in pretreatment, high in detection speed, wide in coverage range and suitable for rapid screening and quality monitoring of pesticide and veterinary drug residues in raw milk, and has good practicability and popularization value.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a non-targeted screening method based on LC-TOFMS for simultaneously screening multiple pesticide and veterinary drug residues in raw milk and its application. Background Technology

[0002] Currently, pesticide and veterinary drug residues in raw milk are significant factors affecting dairy product safety. Most common detection methods are targeted, leading to low efficiency due to complex pretreatment processes, narrow detection ranges, and long processing times. Furthermore, different drug classes require different pretreatment methods and instrument conditions, making rapid and comprehensive screening difficult. With increasing public concern about dairy product safety, developing a non-targeted detection method capable of simultaneously screening for multiple pesticide and veterinary drug residues is of significant practical importance, enabling efficient monitoring of pesticide and veterinary drug residues in raw milk. Summary of the Invention

[0003] The purpose of this invention is to provide a non-targeted rapid screening method for pesticide and veterinary drug residues in raw milk based on LC-TOFMS, establishing a supporting mass spectrometry database to achieve simultaneous qualitative screening of 46 pesticides and / or 54 veterinary drugs in raw milk. This method features simple pretreatment, fast analysis speed, and broad coverage, making it suitable for rapid screening and quality control of pesticide and veterinary drug residues in raw milk.

[0004] In a first aspect, this invention provides a non-targeted detection method for simultaneously screening multiple pesticide and / or veterinary drug residues in raw milk. The method is based on liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS / MS) and comprises the following steps:

[0005] (1) The QuEChERS method was used to pretreat raw milk samples;

[0006] (2) The pretreated sample was injected into a liquid chromatography-time-of-flight mass spectrometer for detection;

[0007] The liquid chromatography conditions are as follows:

[0008] Chromatographic column: 1.7μm C18100A (2.1mm×100mm, 1.7μm); Mobile phase: Phase A is an aqueous solution containing 10mmol / L ammonium formate (containing 0.1% formic acid), Phase B is acetonitrile + methanol = 4:1 (containing 0.1% formic acid); Elution gradient: 0-3min, 5% B-15% B; 3-8min, 15% B-40% B; 8-12min, 40% B-95% B; 12.10-15min, hold at 5% B. Flow rate: 0.3mL / min. Column temperature: 35℃;

[0009] The LC-TOFMS mass spectrometry conditions include: an electrospray ion source; a capillary voltage of 4000V and a nozzle voltage of 300V in positive ion mode; a capillary voltage of 3500V and a nozzle voltage of 1200V in negative ion mode; a fragmentation voltage of 150V and a capillary end voltage of 65V.

[0010] (3) Match the detection data with a pre-established mass spectrometry database to achieve non-targeted qualitative identification of pesticide and veterinary drug residues; the mass spectrometry database contains primary and secondary mass spectrometry information of pesticides and / or veterinary drugs.

[0011] The mass spectrometry database also includes the compound name, molecular formula, CAS number, parent ion loading method, retention time, and fragmentation voltage of pesticides and / or veterinary drugs.

[0012] Furthermore,

[0013] The mass spectrometry database contains mass spectrometry data for at least 46 pesticides and / or at least 54 veterinary drugs;

[0014] The pesticides mentioned include one or more of the following: methamidophos, methomyl, acephate, cymoxanil, carbendazim, omethoate, bifenthrin, carbofuran, acetamiprid, dimethoate, 3-carbofuran, ethoprophos, chlorpyrifos, thiamethoxam, imidacloprid, pendimethalin, tebuconazole, cyproconazole, methamidophos, thiamethoxam, phorate, methyl isofenphos, chlorpyrifos, triazophos, flusilazole, tebuconazole, flutriafol, chlorpyrifos, tebuconazole, cyproconazole, propiconazole, chlorpyrifos, etoxazole, pyridaben, flumorpholine, profenofos, pyraclostrobin, dimethomorph, difenoconazole, abamectin B1a, fipronil, fipronil sulfone, fipronil sulfoxide, flufenoxuron, and cyazofamid.

[0015] The veterinary drugs mentioned include acesulfame, sulfapyridine, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfamethylpyrimidine, sulfadimethoxazole, sulfamethazine, sulfabenzoyl, sulfauracil, sulfadiazine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfachlorpyridazine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfabenzopyrazole, phthalimide, oxaquinic acid, flumethylquine, norfloxacin, enoxacin, ciprofloxacin, pefloxacin, dafloxacin, enrofloxacin, and ofloxacin. One or more of the following: methabolic floxacin, sarafloxacin, difluorofloxacin, clenbuterol, ractopamine, salbutamol, cimaterol, zippaterol, chlorpheniramine, cibuterol, mabuterol, brombuterol, bambuterol, clenbuterol, tolobuterol, ritodrine, clenbuterol, masperidone, clenbuterol, hydroxymethyl clenbuterol, hydroxymetronidazole, metronidazole, dimetridazole, florfenicol, spiramycin, tylosin, erythromycin, roxithromycin, lincomycin, tilmicosin, chloramphenicol, thiamphenicol, and chlorpheniramine.

[0016] The QuEChERS preprocessing steps include:

[0017] Weigh 5g of raw milk sample and add 10mL of acetonitrile for vortex extraction;

[0018] Add commercially available QuEChERS salt extraction kits and centrifuge by shaking.

[0019] The supernatant was purified through a purification tube, filtered, and then injected into the sample.

[0020] Preferably,

[0021] The mass spectrometry databases for the 46 pesticides and 54 veterinary drugs are detailed in Tables 1 and 2.

[0022] A second aspect of the present invention provides a mass spectrometry database applicable to the above-described method: the mass spectrometry database contains mass spectrometry data of at least 46 pesticides and / or 54 veterinary drugs as described above; the mass spectrometry data includes the name, molecular formula, CAS number, parent ion loading method, retention time and fragmentation voltage parameters of the pesticides and / or veterinary drugs, and is stored in the mass spectrometry system's supporting software;

[0023] The 46 pesticides include methamidophos, methomyl, acephate, cymoxanil, carbendazim, omethoate, bifenthrin, carbofuran, acetamiprid, dimethoate, 3-carbofuran, ethoprophos, chlorpyrifos, thiamethoxam, imidacloprid, pendimethalin, tebuconazole, cyproconazole, methamidophos, thiamethoxam, phorate, methyl isofenphos, chlorpyrifos, triazophos, flusilazole, tebuconazole, flutriafol, chlorpyrifos, tebuconazole, cyproconazole, propiconazole, chlorpyrifos, etoxazole, pyridaben, flumorpholine, profenofos, pyraclostrobin, dimethomorph, difenoconazole, abamectin B1a, fipronil, fipronil sulfone, fipronil sulfoxide, flufenoxuron, and cyazofamid (CCIM).

[0024] The 54 veterinary drugs include acesulfame potassium, sulfapyridine, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfamethylpyrimidine, sulfadimethoxazole, sulfamethazine, sulfabenzoyl, sulfauracil, sulfadiazine, sulfamethoxypyrimidine, sulfamethoxypyridazine, sulfaparamethoxypyrimidine, sulfachlorpyridazine, sulfao-dimethoxypyrimidine, sulfa-m-dimethoxypyrimidine, sulfabenzylpyrazole, phthalimide, oxaquinic acid, flumethylquine, norfloxacin, enoxacin, ciprofloxacin, pefloxacin, dafloxacin, and enrofloxacin. Ofloxacin, Mebofloxacin, Sarafloxacin, Difloxacin, Clenbuterol, Ractopamine, Salbutamol, Cimaterol, Zipaterol, Chlorpheniramine, Cibuterol, Mabuterol, Brobuterol, Bambuterol, Clenbuterol, Tuloterol, Ritodrine, Clenbuterol, Mapenterol, Clenbuterol, Hydroxymethyl Clenbuterol, Hydroxymetronidazole, Metronidazole, Dimetridazole, Florfenicol, Spiramycin, Tylosin, Erythromycin, Roxithromycin, Lincomycin, Tilmicosin, Chloramphenicol, Thiamphenicol, Chlorpheniramine.

[0025] Furthermore, the mass spectrometry database can be continuously expanded by adding mass spectrometry data of pesticide or veterinary drug standards.

[0026] During the database construction process, the neutral group loss method was used to identify the parent ions for methamidophos and methyl isofenphos.

[0027] A third aspect of the present invention provides the application of the method described above and the mass spectrometry database described above in the screening and detection of veterinary drug residues and / or pesticide residues.

[0028] Preferably, the above-described method or mass spectrometry database is used for the simultaneous and rapid screening of multiple pesticide and / or veterinary drug residues in raw milk.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. The pretreatment of the method of the present invention is simple and the detection speed is fast (the processing time for a single sample does not exceed 15 minutes and the detection time does not exceed 30 minutes), making it suitable for batch screening;

[0031] 2. The database of this invention has a wide coverage and supports the simultaneous identification of multiple pesticides and veterinary drugs;

[0032] 3. Highly scalable, the database can be continuously expanded by adding standard products;

[0033] 4. Suitable for rapid qualitative screening, providing targeted guidance for subsequent quantitative analysis;

[0034] 5. It has broad application prospects in food safety supervision and dairy product quality control; it provides efficient technical support for monitoring the quality and safety of raw milk. Attached Figure Description

[0035] Figure 1 The distribution diagram of [M+Na]+EIC and ionic isotopes of methamidophos;

[0036] Figure 2 The distribution diagram of methamidophos [M-C3H9N+H]+EIC and its isotopes;

[0037] Figure 3 The molecular structure of methamidophos is shown below;

[0038] Figure 4 The molecular structure of methyl isofenphos isothionylphosphonate;

[0039] Figure 5 This is the primary mass spectrum (EIC) and isotopic ion distribution diagram of methyl isofenphos. Detailed Implementation

[0040] 1. Materials and Methods

[0041] 1.1 Instruments and Reagents

[0042] Instruments: Agilent 1290 ultra-high pressure liquid chromatograph; Agilent 6530 liquid chromatography-time-of-flight mass spectrometer; Thermo Fisher Scientific Heraeus Multifuge X1R high-speed refrigerated centrifuge; Mohr EH2O ultrapure water system; Mettler MA104E 0.001 g electronic balance; Pruibon DMT-2500 multi-tube vortex mixer.

[0043] Reagents: Acetonitrile (HPLC grade, Thermo Fisher Scientific); Formic acid (HPLC grade, Shanghai Anpu); Ammonium formate (AR, Shanghai Husheng); QuEChERS pesticide residue extraction kit and purification tubes from DouDian Biotechnology; QuEChERS veterinary drug residue extraction kit and purification tubes from DouDian Biotechnology; Standard solutions for various pesticide residues (single standard, 1000 μg / ml, Ministry of Agriculture and Rural Affairs Environmental Protection Research and Monitoring Institute, Tanmo Quality Inspection); Standard solutions for amide alcohols (single standard, 100 μg / ml, Tanmo Quality Inspection); Standard solutions for quinolones (single standard, 100 μg / ml, Tanmo Quality Inspection); Standard solutions for sulfonamides (single standard, 100 μg / ml, Tanmo Quality Inspection); Standard solutions for tetracyclines (single standard, 100 μg / ml, Tanmo Quality Inspection); Standard solutions for β-agonists (single standard, 100 μg / ml, Tanmo Quality Inspection); Fresh raw milk from local sources (purchased from a supermarket).

[0044] 1.2 Instrument Conditions

[0045] 1.2.1 Liquid Chromatography Conditions: Column: 1.7μm C18100A column (2.1mm×100mm, 1.7μm); Mobile phase: Phase A is an aqueous solution containing 10mmol / L ammonium formate (containing 0.1% formic acid), Phase B is acetonitrile + methanol = 4:1 (containing 0.1% formic acid); Elution gradient: 0-3 min, 5% B to 15% B; 3-8 min, 15% B to 40% B; 8-12 min, 40% B to 95% B; 12.10-15 min, hold at 5% B. Flow rate: 0.3mL / min. Column temperature: 35℃, Injection volume: 2μL.

[0046] 1.2.2 Time-of-flight mass spectrometry conditions

[0047] The ion source was an electrospray ionization source with jet ion focusing (DualAJS ESI+). The ion source dry gas temperature (GasTemp) was 300℃, the drying gas flow rate (Drying Gas) was 5 L / min, the nebulizer pressure (Nebulizer) was 35 psi, the sheath gas temperature (Sheath Gas Temp) was 350℃, and the sheath gas flow rate (Sheath Gas Flow) was 11 L / min. The primary mass spectrometer fragmentation voltage (Fragmentor) was 150 V, and the skimmer voltage (Skimmer) was set to 65 V. In positive ion mode, the capillary voltage (VCap) was set to 4000 V, and the nozzle voltage (Nozzle Voltage) was set to 300 V; in negative ion mode, the capillary voltage (VCap) was set to 3500 V, and the nozzle voltage (Nozzle Voltage) was set to 1200 V.

[0048] 1.3 Database Establishment

[0049] Using Agilent PCDL Manager software, create a new template and input the names, molecular formulas, and CAS numbers of each pesticide and veterinary drug into the software and save it (separate templates for pesticide and veterinary drug residues). Then, prepare 1 mg / L standard working solutions for each pesticide and veterinary drug residue using acetonitrile. Use Agilent time-of-flight mass spectrometry (TOF-MS) in MSonly (primary mass spectrometry) mode to detect each standard working solution. Use Agilent Qualitative Analysis software to analyze the detected primary mass spectra. Use the Findby Formul function in Target / Suspect Screening of the software to set the ion loading mode: positive ion mode is +H or +NH4, negative ion mode is -H or +HCOO. Select the newly created pesticide and veterinary drug databases as the database sources. Combine the peak response values ​​with the assigned fractions of the searched compounds (mainly by matching isotope distribution and precise relative atomic mass) to determine the retention time of the compounds and fill it into the database. Then, using the Agilent targeted MS / MS (target compound secondary mass spectrometry) mode, input the target compound precursor ion mass-to-nucleus ratio (accurate to 4 decimal places; to ensure accuracy, the molecular formula and loading method can be entered in the Agilent Isotope Distribution Calculator software to confirm the precise value of the precursor ion mass-to-nucleus ratio, or the value can be calculated directly by the software), retention time, and fragmentation voltage (CE) gradient value (set range 5-100V, step size 5V; after determining a suitable range, the step size can be accurate to 1-2V). Other parameters use default values. In this mode, 1 mg / L of each standard solution was detected to obtain the compound's secondary mass spectrum. The Findby Formul function in Target / Suspect Screening was used again to find the secondary mass spectrum for the corresponding compound, and the most suitable fragmentation voltage (CE) value was selected. To ensure non-targeted screening coverage and detection efficiency, mass spectrometry conditions such as DP value and drying gas temperature, which cannot have different values ​​simultaneously in the same method, were not optimized. The parameters of each pesticide residue compound are shown in Table 1, and the parameters of each veterinary drug residue compound are shown in Table 2. Then, a unified targeted MS / MS (target compound secondary mass spectrometry) method was established. All pesticide and veterinary drug residue compound parameters were input into the newly established method (established separately in positive and negative ion modes). The standard working solutions of each pesticide and veterinary drug residue at different concentrations of 1-200 μg / kg were then analyzed again. The secondary spectra of each compound at each concentration were sent to the pesticide and veterinary drug residue database as a database for sample detection.

[0050] Table 1. Mass spectrometry parameters of 46 pesticide residue compounds

[0051]

[0052] Table 2. Mass spectrometry parameters of 54 veterinary drug residue compounds

[0053]

[0054] 1.4 Sample Pretreatment

[0055] Accurately weigh 5g of raw milk sample into a 50mL centrifuge tube, add 10mL of acetonitrile and vortex for 1min (2000r / min). Add the extraction packet and immediately shake vigorously for 5min (2000r / min). Centrifuge at 4℃ and 10000r / min for 5min. Take 5mL of the supernatant and transfer it to a purification tube. Vortex for 5min (2000r / min) and centrifuge at 5000r / min for 5min. Filter the supernatant through a 0.22μm organic phase microporous membrane and transfer it to a sample vial for instrument analysis.

[0056] 2 Results and Discussion

[0057] 2.1 Problem of loss of neutral groups in parent ions during the establishment of primary spectral libraries

[0058] In establishing a primary mass spectrometry database for pesticide residues, the corresponding [M+H] molecules can be found for most of them based on their molecular formula. + [M+NH4] + Or [MH] － The parent ion, but methamidophos (CAS: 24353-61-5, C 11 H 16 NO4PS), according to [M+H) + The loading method should have resulted in an ion peak with a mass-to-nucleus ratio (m / z) of 290.061, but instead, a very high mass-to-nucleus ratio (m / z) peak of 230.9873 appeared. Analysis using Agilent Qualitative Analysis software, by adjusting the loading method and considering the loss of neutral groups, revealed two possible scenarios: one is [M+Na]... + Formal existence (see) Figure 1 Another type exists by losing a neutral group: [M-C3H9N+H] + Formal existence (see) Figure 2 Both loading methods produced high ion peak response values, and the isotope mass distribution maps showed very high agreement. However, Na ions were absent in both the mobile phase and the standard solution solvent, and the [M-C3H9N+H] structure existed by losing neutral groups. + The ion peak response value is higher than that of [M+Na]. + An order of magnitude, close to the response values ​​of other pesticide compounds. This is based on the compound's structural formula ( Figure 3Analysis suggests that the molecule may have simultaneously lost one isopropyl group and one amino group (see...). Figure 3 Therefore, it is considered to be due to the loss of a neutral group ([M-C3H9N+H)). + This is more in line with the actual situation.

[0059] Methyl isofenphos (CAS: 99675-03-3, C 14 H 22 NO4PS has a similar molecular structure to methamidophos, but with an additional isopropyl group. If the loss of the neutral group is not considered, there is only a [M+H]+ ion peak with a relatively low response value. However, if the loss of the neutral group is considered, a [M-C6H]+ ion peak will form. 15 N] + and [M-C3H9N+H] + Two ion peaks with relatively high response values ​​(see) Figure 5 Based on the molecular structure of methyl isofenphos, it can be inferred that an isopropylamine group has been lost, forming [M-C6H]. 15 N] + The parent ion, having simultaneously lost both isopropylamino and isopropyl groups, forms [M-C3H9N+H]. + When performing targeted MS / MS (secondary mass spectrometry) detection of the target compound, one or both of the precursor ions can be selected.

[0060] 2.2 Mutual interference issues during the secondary mass spectrometry detection of target compounds

[0061] During the testing process, some pesticide residues formed [M+H]. + The mass-to-nucleus ratios (m / z) of these compounds are too close, and their retention times are also quite similar; there are also some isomers of animal drug residues, forming [M+H] compounds. +The mass-to-nucleus ratios (m / z) of these compounds are completely identical, and adjusting the mobile phase cannot solve the problem of their excessively close retention times, leading to mutual interference between these two types of compounds during detection. Examples include chlorpyrifos and triazole phosphide, and sulfamethoxypyridazine and sulfamethoxypyrimidine (see Table 3). Although these compounds can be separated during secondary library construction, due to their structural similarity, the highest-response fragment ions are also the same. Therefore, during the validation of spiked recovery, adding only one compound will still cause the analysis software to identify both. While lowering the default retention time bias (Delta Ret.Time) can avoid interference, it reduces the number of compound sampling points, thus lowering the response value. Because the fragment ions of the second strongest response values ​​of these compounds differ, and the distribution ratio of the major fragment ions is roughly fixed, in practical applications, these compounds can be labeled. When such compounds are detected, the type of compound can be determined by comparing the detected secondary mass spectrum with the spectral library.

[0062] Table 3. Secondary mass spectrometry parameters of compounds with similar mass-to-nucleus ratios (m / z)

[0063]

[0064] 2.3 Method Validation

[0065] Minimum Identification Concentration Validation

[0066] The minimum identifiable level of individual pesticide and veterinary drug residues was verified by spiked recovery. Forty-six pesticide residues were prepared as mixed standard working solutions at 1 mg / L using acetonitrile. These mixed standards were then added to raw milk samples to form spiking concentrations of 0.1 mg / kg, 0.05 mg / kg, 0.02 mg / kg, 0.01 mg / kg, 0.005 mg / kg, 0.002 mg / kg, and 0.001 mg / kg, respectively. These solutions, along with unspiked raw milk samples, were extracted and purified using a QuEChERS pesticide residue extraction kit and purification tubes according to method 1.4, and then analyzed using a pre-established unified method in UPLC-QTOFMS. The detected spectra were obtained using Agilent Qualitative Analysis software. The established secondary mass spectrum database of pesticide residues was imported, and the Find by Formul function in Target / Suspect Screening of the software was used to set the ion loading mode (based on the detection results of the standard solution, the positive ion mode only added +H and +NH4 loading modes, and the neutral group loss mode only set -C6H15N and -C3H9N; the negative ion mode only set the -H loading mode, and did not set the neutral group loss mode). The lowest recognizable concentrations of each pesticide residue compound obtained by comparing with the secondary mass spectrum data and eliminating interference terms are shown in Table 4.

[0067] Table 4 Minimum Identification Concentrations and Limits for Pesticide Residues

[0068]

[0069]

[0070] Each veterinary residue standard stock solution was prepared with acetonitrile to form a 1 mg / L mixed standard working solution of 54 veterinary residues. The mixed standard was added to raw milk samples to form spiking concentrations of 200 μg / kg, 100 μg / kg, 50 μg / kg, 20 μg / kg, 10 μg / kg, 5 μg / kg, 2 μg / kg, and 1 μg / kg, respectively. The raw milk samples, together with those without spiking, were extracted and purified using a QuEChERS veterinary residue extraction kit and purification tubes according to method 1.4. The purified samples were then analyzed using a pre-established unified method in UPLC-QTOFMS. The detected spectra were obtained using Agilent Qualitative Analysis software. The established secondary mass spectrum database of pesticide residues was imported, and the Find by Formul function in Target / Suspect Screening of the software was used to set the ion loading mode (based on the detection results of the standard solution, the positive ion mode only added the +H loading mode and did not set the neutral group loss mode; the negative ion mode only set the -H loading mode and did not set the neutral group loss mode). The lowest recognizable concentrations of each pesticide residue compound obtained by comparing with the secondary mass spectrum data and eliminating interference terms are shown in Table 5.

[0071] Table 5 Minimum Identification Concentration and Limit Values ​​for Veterinary Drug Residues

[0072]

[0073]

[0074] 2.4 Advantages of this method

[0075] Based on the minimum identification concentration (MRC) verification results, all tested pesticide residues were below the limits specified in GB 2763-2021 for maximum residue limits of pesticides in food. The vast majority of tested veterinary drug residues were also below the limits specified in GB 31650-2019 and GB31650.1-2022 for maximum residue limits of pesticides in food. The core advantages of this method are: highly efficient and rapid non-targeted screening capability, and accurate detection capability of m / z values ​​based on time-of-flight mass spectrometry. Through continuous database improvement, this method can continuously expand the range of veterinary and pesticide residues screened, covering the vast majority of agricultural and veterinary drug compounds. The pretreatment method is simple, with the pretreatment process for a single sample taking no more than 15 minutes. Because no standard solution is required, the detection of a single sample takes no more than 30 minutes. Using the same liquid chromatography conditions and eliminating the need for reagent preparation during pretreatment saves significant preparation time, ensuring rapid detection of pesticide and veterinary drug residues in samples. It is particularly suitable for accurately screening milk for excessive levels of pesticides and veterinary drugs before quantitative analysis, providing strong technical support for food safety supervision and production quality control.

[0076] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A non-targeted detection method for simultaneous screening of multiple pesticide and / or multiple veterinary drug residues in raw milk, characterized in that, A non-targeted detection method for simultaneously screening a plurality of pesticide and veterinary drug residues in raw milk based on liquid chromatography-time-of-flight mass spectrometry technology, which comprises the following steps: (1) The QuEChERS method is used for pretreatment of the raw milk sample; (2) The pretreated sample is injected into a liquid chromatography-time-of-flight mass spectrometry instrument for detection; The liquid chromatography conditions are as follows: Chromatography column: 1.7 pm C18 100 A (2.1 mm x 100 mm, 1.7 pm); mobile phase: phase A: 10 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid), phase B: acetonitrile + methanol = 4:1 (containing 0.1% formic acid), elution gradient: 0-3 min, 5% B-15% B; 3-8 min, 15% B-40% B; 8-12 min, 40% B-95% B; 12.10-15 min, 5% B is maintained; flow rate: 0.3 mL / min; The column temperature is 35 DEG C. The mass spectrometry conditions of the LC-TOFMS include: the ion source is an electrospray ion source; the capillary voltage in the positive ion mode is 4000V, and the nozzle voltage is 300V; the capillary voltage in the negative ion mode is 3500V, and the nozzle voltage is 1200V; the fragmentation voltage is 150V, and the capillary end voltage is 65V; (3) The detection data is matched with a pre-established mass spectrometry database to realize non-targeted qualitative identification of pesticide and veterinary drug residues; the mass spectrometry database contains the first and second mass spectrometry information of pesticides and veterinary drugs.

2. The method of claim 1, wherein: The mass spectrometry database also includes the compound name, molecular formula, CAS number, parent ion plus mode, retention time, and fragmentation voltage of the pesticide and veterinary drug.

3. The method of claim 1, wherein: The mass spectrometry database contains mass spectrometry data of at least 46 pesticides and / or at least 54 veterinary drugs; The pesticides include one or more of methamidophos, methomyl, acephate, propamocarb, carbendazim, oxydemeton-methyl, bifenthrin, carbofuran, acetamiprid, dimethoate, 3-carbofuran, phenthoate, forchlorfenuron, clothianidin, imidacloprid, pendimethalin, penconazole, myclobutanil, isocarbophos, thiamethoxam, phoxim, phosmet, phorate, trichlorphon, triazophos, flusulfamide, diniconazole, flutriafol, sulprofos, penthrzole, fenbuconazole, propiconazole, chlorpyrifos, etoxazole, pyridaben, flumorph, profenofos, pyraclostrobin, dimethomorph, difenoconazole, abamectin B1a, fipronil, fipronil sulfone, fipronil sulfoxide, flumethrin, and cyprodinil CCIM; The veterinary drugs include one or more of acetylsulfamine, sulfapyridine, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfamethazine, sulfisoxazole, sulfamethazine, sulfabenzamide, sulfamerazine, sulfadimethazine, sulfamonomethoxine, sulfamethoxypyridazine, sulfachlorpyridazine, sulfadimethoxine, sulfamonomethoxine, sulfachlorpyridazine, sulfadimethoxine, sulfabenzamide, sulfamerazine, norfloxacin, enoxacin, ciprofloxacin, perfloxacin, danofloxacin, enrofloxacin, ofloxacin, marbofloxacin, sarafloxacin, difloxacin, clenbuterol, ractopamine, salbutamol, cimaterol, zilpaterol, clorprenaline, cibuteroi, mapenterol, brombuterol, bambuterol, clenbuterol, tulobuterol, ritodrine, clenbuterol, mapenterol, clenbuterol, hydroxymethyl clenbuterol, hydroxymetronidazole, metronidazole, dimetridazole, flunarinic acid, spiramycin, tylosin, erythromycin, roxythromycin, lincomycin, tilmicosin, chloramphenicol, thiamphenicol, and chlorphenicol.

4. The method of claim 1, wherein, The QuEChERS pretreatment step comprises: 5g of the raw milk sample is weighed and added with 10mL of acetonitrile for vortex extraction; Add commercial QuEChERS extraction salt package and shake centrifugation; Take the supernatant and purify through a purification tube, then filter and inject.

5. The method of claim 3, wherein, The mass spectrometry database of the 46 pesticides and 54 veterinary drugs comprises: Table 1 Mass spectrometry parameters of 46 pesticide residue compounds Table 2 Mass spectrometry parameters of 54 veterinary drug residue compounds 6. A mass spectral database for use in the method of any one of claims 1 to 5, characterized in that: The mass spectrometry database comprises at least mass spectrometry data of the 46 pesticides and / or 54 veterinary drugs according to claim 5; the mass spectrometry data comprises the name, molecular formula, CAS number, parent ion adding mode, retention time and fragmentation voltage parameters of the pesticide and / or veterinary drug, and is stored in the software of the mass spectrometry system; The 46 pesticides comprise methamidophos, methomyl, acephate, propamocarb, carbendazim, oxydemeton-methyl, bifenthrin, carbofuran, acetamiprid, dimethoate, 3-carbofuran, phorate, forchlorfenuron, clothianidin, imidacloprid, pendimethalin, penconazole, myclobutanil, isocarbophos, thiamethoxam, phoxim, phosmet, phorate, phenthoate, triazophos, tridemorph, enilconazole, cyproconazole, sulprofos, fipronil, fipronil sulfone, fipronil sulfoxide, flumethrin, cyazofamid CCIM; The 54 veterinary drugs comprise acetylsulfamine, sulfapyridine, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfamethazine, sulfisoxazole, sulfamethazine, sulfabenzamide, sulfamerazine, sulfadimethazine, sulfamonomethoxine, sulfamethoxypyridazine, sulfadimethoxine, sulfachlorpyridazine, sulfachrysoidine, sulfamonomethoxine, sulfabenzpyrazone, sulfathalidine, ormetoprim, flumequine, norfloxacin, enoxacin, ciprofloxacin, perfloxacin, danofloxacin, enrofloxacin, ofloxacin, marbofloxacin, sarafloxacin, difloxacin, clenbuterol, ractopamine, salbutamol, cimaterol, zilpaterol, clorprenaline, cibutered, mapenterol, brombuterol, bambuterol, clenbuterol, tulobuterol, ritodrine, clenbuterol, mapenterol, hydroxymethyl clenbuterol, hydroxymetronidazole, metronidazole, dimetridazole, flunarinic acid, spiramycin, tylosin, erythromycin, roxythromycin, lincomycin, tilmicosin, chloramphenicol, thiamphenicol, chlorphenicol.

7. The mass spectral database of claim 6, wherein: The mass spectrometry database is continuously expanded by adding mass spectrometry data of standard samples of pesticides or veterinary drugs.

8. The mass spectral database of claim 6, wherein: In the database construction process, the neutral group loss mode is used for parent ion recognition for isocarbophos and phosmet.

9. The method according to any one of claims 1-5 and the mass spectrometry database according to any one of claims 6-8 are used in the screening detection of veterinary drug residues and / or pesticide residues.

10. Use according to claim 9, characterized in that: The method is used in the simultaneous rapid screening of multiple pesticide residues and / or multiple veterinary drug residues in raw milk.

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