CN105699565B - Detect the method and liquid matter database of left drug in animal-derived food - Google Patents

Abstract

The present invention relates to a kind of liquid matter database and its application method for being used to detect left drug in animal-derived food, the preparation of the database comprises the following steps：(1) preparation of standard working solution；(2) pre-treatment is analyzed, sample to be tested analysis pre-treatment work is carried out using the extraction and cleaning technology of rapid enzymolysis+quick SPE (SPE)；(3) disposable sample introduction chromatography；(4) liquid mass spectral database is built.

Description

Method for detecting residual medicine in animal-derived food and liquid-mass database

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a liquid quality database for detecting residual medicines in animal-derived food and a using method thereof.

Background

Animal-derived food accounts for a considerable proportion in international agricultural product trade, the regulation and restriction on drug residues in animal-derived food are more and more strict, taking a 'positive list' system implemented in 5 months in 06 years in Japan as an example, a specific limit standard is set for 236 veterinary drugs and feed additives, and in the field of domestic food safety, from early chloromycetin and nitrofuran to later malachite green and clenbuterol, the frequent occurrence of food safety events caused by drug residues highlights the technical support capacity insufficiency of a food safety supervision and control system in China, and a rapid screening method for various residues is urgently needed to be established. At present, the detection technology and method for drug residues in animal-derived food are developed from single-residue analysis to single-type multi-residue analysis, and the multi-residue detection standard classified by the species of substances accounts for more than 70% of national and industrial standards of residue analysis in 03 years. Meanwhile, mass spectrometry has become a main technical means for multi-residue detection, and the technical index rule based on the european union 2002/657/EC regulation as the blue book is also internationally recognized and accepted. With the development of the residual analysis technology, the multi-type residual analysis has advantages in various aspects such as information quantity, detection efficiency and the like, so that the multi-type residual analysis becomes a research and development hotspot, and the practicability of the tandem mass spectrometers in the field of mass spectrometry equipment such as Q-TOF, Q-Trap and the like provides technical possibility for the rapid analysis of the multi-type residual through the gradual maturity of spectral library comparison based on the advanced mass spectrometry technology. However, the simultaneous detection of various types of residues, whether in terms of process development, validation and standardization or in terms of regulatory perfection, presents major difficulties:

(1) the physicochemical properties of different veterinary drugs are greatly different, the polarity coverage is wide, the residual forms in different matrixes are different, and individual veterinary drugs need to be effectively detected by a derivatization party, so that effective breakthrough is difficult to realize in a general pretreatment method and chromatographic one-time sample injection analysis;

(2) the regulation has different limit requirements and use regulations on different veterinary drugs (forbidden drugs and limited drugs), and the multi-type residue detection and verification rules are not complete;

(3) blank matrixes required by method development are difficult to obtain, and mixed standard solutions are difficult to prepare;

(4) analytical capabilities of the instrument. In terms of constructing a liquid mass spectrum library, there are commercial spectrum libraries such as a forensic toxicant database (1250 target analytes) of AB corporation, a veterinary drug database (139 target analytes); small molecule drug database at Freiburg university hospital, etc., but there are the following application limitations:

1) only MS/MS data is available, and no spectrum system information characteristic is available;

2) no online information feature;

3) there is no coupling of uniform pretreatment method and instrument analysis;

4) matrix standard data is lacking.

Therefore, under the existing conditions, it is difficult to effectively realize the rapid screening and analysis of various residues based on the liquid mass spectrometry library technology.

Disclosure of Invention

In view of the technical difficulties of sample universality pretreatment, rapid analysis of instruments, screening method verification and the like in the analysis of various residual drugs of animal-derived food at home and abroad at present. The invention aims to establish a rapid screening detection system for multi-species residues of high-risk drugs in animal-derived food, realizes effective reservation and separation of multi-target analyte wide polarity range liquid chromatography by adopting multi-solvent system sectional combination extraction, establishing a high-throughput universal pretreatment technology of samples and comprehensively optimizing a chromatography system, breaks through the difficulty of multi-species residue analysis technology by establishing a liquid chromatography-mass spectrum/mass spectrum database covering multi-dimensional information of chromatography-mass spectrum and carrying out research and development of key technical contents such as screening method verification according to regulatory requirements and detection practice, and establishes an open rapid analysis platform and an effective screening detection technology system which take the multi-species residue universal pretreatment technology, the rapid separation system and the liquid mass spectrum database as technical characteristics.

The invention aims at 103 high-risk residual medicines (B-type residual substances) with the limit value of more than 1.0 mu g/kg as detection targets, the group of medicines are shown in a table 1,

TABLE 1 target analyte chemistry information

The invention firstly relates to a construction method of a rapid screening liquid mass spectrum library for detecting various residues of high-risk drugs in animal-derived food, wherein the animal-derived food is poultry egg food or honey food, and concretely comprises the following steps,

(1) preparing a standard working solution;

a. androgen substance mixed standard stock solution (0.01 g/L): comprises trenbolone, methyltestosterone, nandrolone propionate, nandrolone phenylpropionate, testosterone propionate, dehydrotestosterone, and dehydromethyltestosterone, and the preparation solvent is acetonitrile;

b. progestogen substance mixed standard stock solution (0.01 g/L): comprises medroxyprogesterone, medroxyprogesterone acetate, megestrol, acetate progesterone, melengestrol and acetate 17 alpha-hydroxyprogesterone, and the preparation solvent is acetonitrile;

c. glucocorticoid substance mixed standard stock solution (0.01 g/L): comprises beclomethasone, fludrocortisone acetate, hydrocortisone, methylprednisolone, prednisone and cortisone, and the preparation solvent is methanol;

mixed standard stock solution (0.01g/L) of 5-nitroimidazole drugs and metabolites thereof: comprises ipronidazole, metronidazole, hydroxymetronidazole and hydroxyipronidazole, and the preparation solvent is acetonitrile;

e. nitrofuran metabolites and 2-nitrobenzaldehyde-derivatized nitrofuran metabolites mixed standard stock solutions (0.01 g/L): furacilin metabolite, nitrofurantoin metabolite, furazolidone metabolite, furaltadone metabolite and their respective 2-nitrobenzaldehyde derivatives, the preparation solvent is acetonitrile;

f. fluoroquinolone substance mixed standard stock solution (0.01 g/L): comprises sarafloxacin, enrofloxacin, ciprofloxacin, ofloxacin, norfloxacin, lomefloxacin, pefloxacin, sparfloxacin, difloxacin, danofloxacin, marbofloxacin, orbifloxacin, enoxacin and flumequine, and the preparation solvent is acetonitrile;

g. quinolones mix standard stock solution (0.01 g/L): comprises pipemidic acid, oxolinic acid, pyrrole acid and nalidixic acid, and the preparation solvent is acetonitrile;

h. beta-receptor agonist class of substance mixed standard stock solution (0.01 g/L): comprises salbutamol, terbutaline, ractopamine, salmeterol, fenoterol, methoxytyramine, chlorpropaline and penbutolol, and the preparation solvent is acetonitrile;

standard stock solutions of sulfonamides and synergists thereof mixed (0.01 g/L): comprises sulfadiazine, sulfanilamide-5- (p) trimethoprim, sulfadimidine, sulfaquinoxaline, sulfadimethoxine, sulfanilamide-6- (m) trimethoprim, sulfamethoxypyridazine, sulfamethoxazole, sulfathiazole, sulfacetamide, sulfamethoxazole, sulfamethizole, sulfachloropyridazine, sulfadimidine, sulfapyridine, sulfaphenazole, sulfachloropyrazine, sulfamethazine, sulfanitrobenzene, sulfaphenacyl, sulfa-o-dimethoxypyrimidine, sulfanilamide, sulfaguanidine and trimethoprim, and the preparation solvent is acetonitrile;

k. macrolide mixed standard stock solutions (0.01 g/L): comprises spiramycin, kitasamycin, tilmicosin, midecamycin, clarithromycin, azithromycin, roxithromycin, josamycin, tylosin, oleandomycin and erythromycin, and the preparation solvent is acetonitrile;

lincomycin standard stock solution (0.01g/L), and the preparation solvent is acetonitrile;

standard stock solution of tetracycline mixture (0.01 g/L): comprises tetracycline, oxytetracycline, chlortetracycline and doxycycline, and the preparation solvent is acetonitrile;

polyether substance mixed standard stock solution (0.01 g/L): comprises salinomycin, methyl salinomycin, monensin, maduramicin and lasalocid, and the preparation solvent is methanol;

dye class mixed standard stock solution (0.01 g/L): comprises malachite green and leucomalachite green, and the solvent is methanol;

99 substances mix standard working solution (0.5 mg/L): transferring 0.5mL of the mixed standard stock solution (except the mixed standard stock solution of the nitrofuran metabolite and the 2-nitrobenzaldehyde derivative thereof) into a 10mL volumetric flask, fixing the volume to the scale with acetonitrile, and uniformly mixing;

103 substances mix standard working solution (0.5 mg/L): transferring 0.5mL of each mixed standard storage solution (except the mixed standard storage solution of the nitrofuran metabolites) into a 10mL volumetric flask, metering the volume to a scale with acetonitrile, and uniformly mixing;

nitrofuran metabolite mix standard working solution (0.5 mg/L): respectively transferring 0.5mL of nitrofuran metabolite mixed standard stock solution into a 10mL volumetric flask, metering the volume to the scale with acetonitrile, and uniformly mixing.

(2) The pretreatment of analysis, namely, the pretreatment of the sample to be detected by adopting the extraction and purification technology of rapid enzymolysis (releasing combined state residue) + rapid Solid Phase Extraction (SPE), wherein the specific extraction and purification process is as follows:

a) weighing 2g (to the nearest 0.01g) of homogeneous sample, placing in a 50mL Teflon centrifuge tube (if necessary, adding sample, adding mixed standard working solution with appropriate concentration at this step, and standing in the dark for 30min), adding 3.75mL of ammonium acetate buffer (0.2mol/L, pH 5.2), Na₂EDTA-Mclvaine buffer solution 200 μ L, β -glucuronide/sulfatase 50 μ L, vortex mixing, 50 ℃ water bath oscillation for 2h, cooling to room temperature, centrifuging at 10 ℃, 15000rpm for 5min to obtain supernatant as extract A, and reserving for use.5 mL of 1% formic acid acetonitrile solution is added into the centrifugation residue, vortex mixing, 50 ℃ water bath oscillation for 30min, cooling to room temperature, centrifuging at 10 ℃, 15000rpm for 5min, blowing the supernatant at 30 ℃ to 1mL, adding ammonium formate (5mmol/L) -formic acid (0.1%) -aqueous solution 3mL to obtain extract B, reserving for use. A, B μ L of each extract in Eppendorf tube, centrifuging at 4 ℃, 20000rpm for 10min, taking supernatant 400 μ L, adding ammonium formate (5mmol/L) -formic acid (0.1%) -aqueous solution 400 μ L, performing ultrasonic 1min, and passing through 0.22 μ M mass spectrometry, and performing liquid chromatography-tandem microporous membrane assay.

b) Screening furan metabolites by eggs and honey: weighing 2g of uniform sample (accurate to 0.01g), placing the sample in a 50mL polytetrafluoroethylene centrifuge tube, adding 3.8mL of 0.1mol/L hydrochloric acid solution and 200 μ L of 2-nitrobenzaldehyde solution (0.1mol/L) (if a sample is required to be prepared, adding a nitrofuran metabolite working solution with a proper concentration at the step, placing the sample in the dark for 30min, adjusting the volume sum of the added liquid to be 4mL), vortex and mixing the sample evenly, oscillating the sample in a 50 ℃ water bath for 2h, cooling the sample to room temperature, centrifuging the sample at 10 ℃ and 15000rpm for 10min, taking 200 μ L of supernatant, adding 800 μ L of ammonium formate (5mmol/L) -formic acid (0.1%) -aqueous solution, performing ultrasonic treatment for 1min, passing the sample through a 0.22 μ M microporous filter membrane, and performing liquid chromatography-tandem mass spectrometry.

(3) One-time sample introduction chromatographic analysis

The method comprises the following steps of (1) using a Kinetex C18 chromatographic column, acetonitrile as an organic phase, formic acid as an ionization reinforcing agent, formate as a peak shape optimizing agent, and a combination of water phase/acidic acetonitrile as an optimized mobile phase system;

the concrete conditions are as follows: a chromatographic column: kinetex C18,2.6 μm, 2.1mm × 100mm i.d.;

flow rate: 0.2 mL/min;

sample introduction amount: 10 mu L of the solution;

column temperature: 30 ℃;

gradient elution procedure: (A: 0.1% formic acid-acetonitrile solution; B: ammonium formate (5mmol/L) -formic acid (0.1%) -aqueous solution)

0～2min：5％A；

～8min：20％A；

～15min：95％A；

～16min：100％A；

～19min：100％A；

20min：5％A；

(4) Construction of liquid Mass Spectroscopy library

An advanced acquisition mode of a quadrupole/ion trap tandem mass spectrometry is adopted: presetting multi-reaction detection (sMRM) -Information Dependent Acquisition (IDA) -enhancer ion scanning (EPI);

the mass spectrum parameter determination process is as follows: diluting the mixed standard stock solution to a concentration of 0.2mg/L according to the type of the target analyte by using an initial mobile phase, injecting the diluted mixed standard stock solution into a mass spectrometry ion source at a flow rate of 5 mu L/min by using a constant-current injection pump for parameter optimization,

determining parent Ions and daughter Ions of a target analyte by using scanning modes of Q1MS, Q1Multiple Ions, Product Ions, MRM and the like respectively, and optimizing and determining compound parameters such as depolymerization voltage (DP), collision cell inlet voltage (EP), Collision Energy (CE), collision cell outlet voltage (CXP) and the like by using a Ramp function;

mass spectrometry conditions (API 4000 and API 4000Q-TRAP):

a) an ion source: an electrospray ion source;

b) the scanning mode is as follows: scanning positive ions;

c) the detection mode is as follows: sMRM-IDA-EPI

d) Electrospray voltage: 5500V;

e) atomizing gas pressure: 40 psi;

f) air curtain pressure: 30 psi;

g) auxiliary gas pressure: 45 psi;

h) ion source temperature: 475 ℃;

i) sMRM parameter setting: setting an MRM detection window to be 60s, and setting the scanning time of the target object to be 1.4 s;

j) IDA rule: response threshold: 3000 cps; dynamic background subtraction; the strongest ion is selected to be 1 to 3;

k) enhancer ion scan (EPI) parameter settings: the scanning mass number range is 70-1000 Da; the scanning speed is 10000 Da/s; the scanning accumulation frequency is 1; the collision energy was 35 eV; the extended collision energy is 15 eV;

and (3) constructing a liquid mass spectrum library aiming at mass spectrum results of 103B substances.

The library establishing software used in the construction of the database is Analyst1.5 of AB SCIEX company, and the standard of library establishment is set as follows according to the design thought:

(1) giving the RT value of each target analyte under the condition system of the set liquid chromatogram in the research;

(2) giving the relevant chemical information (e.g., name, chemical formula, molecular weight, CAS number, compound class, ID, molecular structural formula, etc.) for each target analyte;

(3) giving at least 5 EPI spectra acquired as described in item 3.2.2 for each target analyte under different conditions, i.e. 4 EPI spectra with a CE of 20eV, 35eV, 50eV and a CE of 35eV and a CES of 15eV, and at least one EPI spectrum with a CE of 35eV and a CES of 15eV under chromatographic conditions;

(4) the EPI spectra of the target analytes in different matrices are given as much as possible.

The first 3 library building criteria are conditions that must be met, and item 4 is an optional criterion according to the actual situation.

The chromatographic system and mass spectrum conditions selected according to the conditions realize the high-efficiency separation of the multiple residues of 115 veterinary drugs containing 103 high-residue B substances. Although the retention time of individual compounds is close, the ion current spectrum extracted by the compounds can realize mass spectrum separation and can be used for quantitative analysis, and in addition, the fragment information characteristics of the target analytes in the EPI spectrum library represent the qualitative structure 'fingerprint' information of the compounds, so that the target analytes can be accurately determined. The liquid mass spectrum library containing the liquid mass information of the B-type residual substances can realize the rapid screening of various residues of high-risk drugs in the animal-derived food.

Under the above chromatographic and mass spectrometric conditions, except 103 species of species B, a total ion flow graph of 115 species of target analytes, a typical selected ion flow graph, an extracted ion flow graph of each target analyte, and a database spectrogram when CES extension (i.e., CE35eV, CES 15eV) is selected are shown in fig. 1 to 4.

The invention also relates to a rapid screening liquid mass spectrum library which is constructed and obtained by the method and used for detecting various residues of high-risk drugs in animal-derived food, wherein the animal-derived food is poultry egg food or honey food;

the high-risk drug is a high-risk residual drug (B-type residual substance) with a limit value of more than 1.0 mu g/kg, and the group of drugs is shown in Table 1.

The invention also relates to application of the rapid screening liquid mass spectrum library for detecting various residues of high-risk drugs in animal-derived food in detection of drug residues in animal-derived food.

The invention also relates to a method for qualitatively or quantitatively analyzing high-risk drug residues in target animal-derived food by using the liquid mass spectrum library, which comprises the following steps,

(1) a standard solution is prepared and then a standard solution is prepared,

(2) pretreatment of analysis, namely, pretreatment of the sample to be detected by adopting an extraction and purification technology of rapid enzymolysis (releasing combined state residue) and rapid solid-phase extraction (SPE),

(3) and carrying out chromatography and liquid quality analysis on the sample to be detected to obtain a liquid quality spectrogram, comparing the liquid quality spectrogram with the liquid quality spectrogram library, and carrying out qualitative or quantitative analysis and detection.

The qualitative analysis is carried out by searching a spectrum library, and the qualitative standards are as follows:

(1) the retention time of the extracted ion stream of the compound in the sample does not vary by more than 5% from the retention time of the extracted ion stream of the target analyte in the standard solution or the added sample.

(2) The parent/daughter ions (transport ion pairs) of the target analyte must occur simultaneously and the signal-to-noise ratio (S/N) of the transport ion pairs is > 3.

(3) And (3) comparing the EPI spectrogram of the compound in the sample with the EPI spectrogram of the standard solution or the matrix standard solution with the similar concentration level (same concentration order) in the spectrogram library, wherein the spectrogram matching Purity (Purity value) is more than or equal to 60.

The quantitative method of the quantitative analysis is as follows:

quantification was performed using an external standard single point method, and the amount of target analyte was calculated as equation (1).

In formula (1):

x-the amount of target analyte in the sample, μ g/kg;

c-concentration of target analyte in sample solution,. mu.g/L;

v, the volume of the sample solution is determined to be mL;

m-mass of sample, g;

r-recovery,%.

Drawings

FIG. 1 shows a total ion flow graph of 115 target analytes, class A and class B.

FIG. 2 is a typical selected ion flow graph of 115 target analytes for class A and class B species.

Fig. 3 shows an ion flow diagram for extracting 115 target analytes, including a type a substance and a type B substance, fig. 3A shows an ion flow diagram-1, and fig. 3B shows an ion flow diagram-2.

FIG. 4 is a database diagram of a target analyte library for class B species, FIG. 4A, library database diagram-1, FIG. 4B, library database diagram-2.

FIG. 53 is a sectional type extraction and sectional determination mass chromatogram and a 3 rd stage extraction ion flow diagram of nitrofuran metabolites.

Detailed Description

EXAMPLE 1 preparation of Standard working solutions

111 materials mix standard working solutions (0.5 mg/L): and (3) transferring 0.5mL of the mixed standard stock solution (except the mixed standard stock solution of the nitrofuran metabolite and the 2-nitrobenzaldehyde derivative thereof) into a 10mL volumetric flask, metering to a scale with acetonitrile, and mixing uniformly. The solution is stable for 1 month at-20 deg.C.

115 substances mixed standard working solution (0.5 mg/L): and (3) transferring 0.5mL of each mixed standard storage solution (except the mixed standard storage solution of the nitrofuran metabolite) into a 10mL volumetric flask, metering the volume to the scale with acetonitrile, and uniformly mixing. The solution is stable for 1 month at-20 deg.C.

Nitrofuran metabolite mix standard working solution (0.5 mg/L): respectively transferring 0.5mL of nitrofuran metabolite mixed standard stock solution into a 10mL volumetric flask, metering the volume to the scale with acetonitrile, and uniformly mixing. The solution is stable for 3 months at-20 deg.C.

Example 2 pretreatment of analysis

For the pretreatment optimization of 103 target compounds (group B substances), the concept of experimental design is as follows: (1) the simpler the pretreatment process, the better, if possible; (2) selecting a eurytopic extraction solvent; (3) the solvent consumption is reduced, and simultaneously the target analyte loss caused by solvent conversion is avoided as much as possible; second, the target analytes and matrices were analyzed as follows: (1) hormone substances and beta-receptor stimulant substances in a target analyte need an enzymolysis process to release binding state residual drugs; (2) the measurement of the nitrofuran metabolites requires acidolysis and derivatization processes; (3) the sample matrix selected was a liquid animal derived product (honey); (4) among the target analytes are forbidden substances and limiting substances.

The specific extraction and purification process is as follows:

a) weighing 2g (to the nearest 0.01g) of homogeneous sample, placing in 50mL polytetrafluoroethylene for centrifugationIn a tube (if necessary, add sample, add appropriate concentration of mixed standard working solution at this step, and leave in the dark for 30min), add 3.75mL of ammonium acetate buffer (0.2mol/L, pH 5.2), Na₂EDTA-Mclvaine buffer solution 200 μ L, β -glucuronide/sulfatase 50 μ L, vortex mixing, 50 ℃ water bath oscillation 2h, cooling to room temperature, centrifuging at 10 ℃, 15000rpm for 5min to obtain supernatant as extract A, and leaving to be used, adding 1% formic acid acetonitrile solution (2.1.2.6)5mL to the centrifugation residue, vortex mixing, 50 ℃ water bath oscillation 30min, cooling to room temperature, centrifuging at 10 ℃, 15000rpm for 5min, blowing the supernatant to 1mL at 30 ℃, adding ammonium formate (5mmol/L) -formic acid (0.1%) -aqueous solution 3mL to obtain extract B, and leaving to be used, adding 0.1mol/L hydrochloric acid solution 3.8mL to the centrifugation residue, 2-nitrobenzaldehyde solution 200 μ L (if preparing nitrofuran metabolite, adding appropriate concentration nitrofuran metabolite mixing standard working solution at this step, and taking supernatant as supernatant after vortex cooling in a vortex chromatography, adding thereto, vortex chromatography using a filter membrane to obtain supernatant, vortex chromatography using a vortex chromatography column (400 μ rpm-400 μ L), and measuring by vortex chromatography using a vortex chromatography column chromatography with a vortex column chromatography (400 μ rpm-400 μ L) and a vortex chromatography (400 μ rpm, 10 μ L) to obtain supernatant C, and a liquid chromatography column chromatography (400 μ L).

b) Screening furan metabolites by eggs and honey: weighing 2g of uniform sample (accurate to 0.01g), placing the sample in a 50mL polytetrafluoroethylene centrifuge tube, adding 3.8mL of 0.1mol/L hydrochloric acid solution and 200 μ L of 2-nitrobenzaldehyde solution (if a sample is required to be prepared, adding a nitrofuran metabolite working solution with a proper concentration in the step, placing the sample in the dark for 30min, adjusting the volume sum of the added liquid to be 4mL), vortex and mixing uniformly, oscillating the sample in a water bath at 50 ℃ for 2h, cooling the sample to room temperature, centrifuging the sample at 10 ℃ and 15000rpm for 10min, taking 200 μ L of supernatant, adding 800 μ L of ammonium formate (5mmol/L) -formic acid (0.1%) -aqueous solution, performing ultrasonic treatment for 1min, passing through a 0.22 μ M microporous filter membrane, and performing liquid chromatography-tandem mass spectrometry.

In view of the above design concept, the pretreatment procedure was determined through experiments. In the pretreatment stepThe ammonium acetate buffer solution is added to provide pH environment for enzymolysis, and Na is added₂EDTA-Mclvaine solutions are used to facilitate the extraction of tetracyclines and macrolides to prevent them from forming chelates with divalent or trivalent metal cations in the tissue or extraction environment^[18]. Acetonitrile was chosen as the extraction solvent in the second stage because: (1) acetonitrile can be mixed and dissolved with water, and is also a wide-ranging extraction solvent^[13](ii) a (2) Compared with common extraction reagents such as methanol, ethyl acetate and the like, the interference of the acetonitrile extracting solution matrix is relatively small; (3) acetonitrile can precipitate proteins; (4) acetonitrile can denature enzymes and reduce the degradation of analytes during extraction. Acidification of acetonitrile with 1% formic acid is the extraction solvent we determined when studying the extraction of sulfa and floxacin multi-residues, while acidification of acetonitrile is an effective solvent for extraction of multi-residues. The concentration of the 1% formic acid acetonitrile extract was carried out mainly for the purpose of adjusting the ratio of the organic phase and the aqueous phase to the starting mobile phase of the chromatography. The third stage is the acid hydrolysis and derivatization of furan metabolites. Because the enzymolysis is carried out before (the first stage), the acidolysis time can be completed in a short time, the derivatization speed is high, the acidolysis and the derivatization can be completed within 30min, and the volume of a derivatization reagent and a hydrochloric acid solution is controlled during the period mainly to ensure the unification of dilution times. The aim of the method finally adopting 20000rpm refrigerated centrifugation is to remove macromolecular substances in the matrix as much as possible, and the volume is mainly adjusted to be uniform dilution times. It is worth to say that for the matrixes such as eggs and honey, solid/liquid separation is not carried out in the second-stage centrifugation, and the screening including the furan metabolites cannot be completed at one time, so the operation of the link b) is designed to complete the screening of the furan metabolites of the matrixes.

In the 3-segment extraction step, the following key points are as follows: (1) influence of enzymolysis conditions on extraction of a target object; (2) whether the furan metabolite in the combined state is dissociated into a free state in the extraction processes of the first section and the second stage is extracted; (3) the effect of acid hydrolysis and derivatization time on the extraction of the target. To determine these several key points, we validated the method using a capacity validation sample (a positive sample obtained after administration to the animal). The experimental design and results are shown in table 8.

Table 83 segment type preprocessing method design verification and result table

^aExcept for the key points listed in the table, the remaining pretreatment processes were carried out as described with reference to 2.3.2.

^bThe recovery rate is obtained by conversion based on the value measured by a CNAL approved method (both target analytes are quantified by an isotope internal standard method, and the recovery rate ranges from 90% to 110%).

^CWithin the target analyte retention time window, no ion flux chromatographic peaks were significantly extracted.

For group B material we expected STC of 0.5. mu.g/kg, we performed a spiked addition at the 0.5. mu.g/kg level using furatanone metabolite positive muscle matrix, and the results were determined separately for the 3-stage extracts, as shown in FIG. 5.

As can be seen in FIG. 5, the sample matrix effect is greater and there are significant matrix interference peaks due to no further freeze high speed centrifugation. But the overall effect of the 3-segment extraction is clearly visible; part of the compound is completely extracted in the first stage, while part of the compound is extracted in both the first and second stages, and in the third stage, there is almost no other target analyte, while furan metabolites are the main target for extraction, and the AMOZ response is high due to the signal superposition with the AMOZ in the mixed standard by using the positive matrix of the AMOZ.

In conclusion, in the pretreatment process of designing and optimizing the group B103 substances, a reasonable extraction process and an operation method for improving the sample flux as much as possible are designed according to different chemical properties and residual characteristics of different target analytes, and a universal sample pretreatment concept of 'high-speed freezing centrifugation technology', 'Dilute and Shoot' is introduced and successfully applied, so that effective pretreatment of the 103 target analytes is realized. The results demonstrate that "Dilute and Shoot" is the simplest sample preparation strategy, and is particularly suitable for the determination of multiple species of residues.

Example 3 study of one-time sample chromatography System

1. Selection of chromatography columns

The liquid chromatographic separation of multi-target analytes usually considers the use of an ultrafine particle (particle size < 2 mu m) chromatographic column at present, but the research adopts a conventional HPLC system, the upper limit of the pressure resistance of the conventional HPLC system is 400Bar, and therefore, the ultra-high performance liquid chromatographic column cannot be used. For the purpose of separation, the particle size range of the chromatographic column is controlled between 2 mu m and 3 mu m in consideration of the pressure limit of the system, and the length range of the chromatographic column is 100mm to 150 mm; in addition, because of the wide variety of compounds and the wide span of polarity ranges, a C18 column suitable for separating a wide polarity range was selected for the type of column. With the above definitions, the study selected one of each class of compounds according to polarity (selected with reference to LogD values), and a total of 4 substances as analytes representative of 4 types of liquid chromatography columns screened. The screening conditions and results are shown in Table 2.

TABLE 2 chromatographic column Selectivity Experimental design and separation analysis results

As can be seen from the above table, the Kinetex C18 column was better in both resolution and sensitivity than the other columns. Although the column has the smallest particle size, the column back pressure is still within the conventional liquid phase pressure resistance range. And the filling particles adopt the advanced core-shell technology, and compared with the traditional completely porous silica gel column, the separation degree and the sensitivity can be obviously improved. Studies have determined that the use of this type of column is the preferred separation column.

2. Selection of mobile phase system and determination of other chromatographic conditions

The mass spectrometry analysis of commonly used mobile phases (water, formic acid-water solution, acetic acid-water solution, methanol, acetonitrile, acidified methanol, acidified acetonitrile, ammonium formate buffer, ammonium acetate buffer, etc.) and compositions using 103 mixed standard working solutions were screened. On the basis of comprehensively considering factors such as separation degree, sensitivity, analysis time and the like, acetonitrile is finally determined to be used as an organic phase, formic acid is used as an ionization reinforcing agent, formate is used as a peak shape optimizing agent, and the combination of water phase/acidic acetonitrile is used as an optimized mobile phase system.

Liquid chromatography parameters such as sample volume, column temperature and flow rate are optimized, and the main consideration factors comprise chromatographic resolution, sensitivity, reproducibility, matrix effect and the like. Chromatographic separation experiments were performed on the screening target concentration level (0.5. mu.g/kg) by using 103 target mixed standard solutions and determining the optimized value.

On the basis of optimizing related parameters and comparing multiple test results, the specific conditions of the disposable liquid chromatography separation system determined by the research are as follows: a chromatographic column: kinetex C18,2.6 μm, 2.1mm × 100mm i.d.; flow rate: 0.2 mL; sample introduction amount: 10 mu L of the solution; column temperature: at 30 ℃. Gradient elution procedure: (A: 0.1% formic acid-acetonitrile solution; B: ammonium formate (5mmol/L) -formic acid (0.1%) -aq.) for 0-2 min: 5% of A; 8 min: 20% of A; 15 min: 95% of A; 16-19 min: 100% of A; 20-35 min: 5% of A.

Example 4 construction of liquid Mass Spectroscopy library-establishment of Mass Spectroscopy Collection method

Establishment of Mass Spectrometry Collection method

The study adopted the high-level acquisition mode of quadrupole/ion trap tandem mass spectrometry: presetting multi-response detection (sMRM) -Information Dependent Acquisition (IDA) -enhancer ion scanning (EPI). The acquisition mode is established by firstly determining the retention time of the target analyte in a chromatographic system, secondly establishing a multi-reaction monitoring (MRM) mass spectrometry method, then setting the condition for triggering EPI acquisition (namely IDA setting), and finally determining the condition setting of the EPI acquisition.

To establish a corresponding mass spectrometry acquisition method, first, mass spectrometry parameters of 103 target analytes are experimentally set. The research adopts the classification to optimize the compound parameters (including parent ions, ionic ions, depolymerization voltage, collision energy and the like) and then selects the substitution table compound to optimize the ion source parameters (including atomization gas pressure, ion source temperature and the like).

Optimizing and determining parameters of the compound. The RT parameters are set according to the chromatographic separation result. The mass spectrum parameter determination process is as follows: the mixed standard stock solution was diluted to a concentration of 0.2mg/L by the target analyte class using the initial mobile phase, but it was noted that target analytes of the same molecular weight in the same class were formulated separately (since the instrument used for the study was standard resolving, isomers could not be distinguished) and then injected into the mass spectrometry ion source using a constant flow syringe pump at a flow rate of 5 μ L/min for parameter optimization. Scanning modes such as Q1MS, Q1Multiple Ions, Product Ions, MRM and the like are respectively used for determining parent Ions of target analytes, daughter Ions (at least 2 pairs of transmission Ions are selected for each target analyte, an optimal pair of Ions are selected as acquisition Ions of the sMRM in subsequent experiments according to the response signal-to-noise ratio and the interference condition in a blank matrix), and compound parameters such as depolymerization voltage (DP), collision cell inlet voltage (EP), Collision Energy (CE), collision cell outlet voltage (CXP) and the like are optimized and determined by using a Ramp function, and the optimization results of 103 compound parameters are shown in Table 3.

TABLE 3103 target analytes optimize mass spectrometry conditions and reference retention times

Note that the retention time of the target analytes is only used as a reference, because the number of compounds is large, the retention times of some target analytes are very close, the retention time varies due to slight changes of mobile phase, and the sequence of individual compounds varies.

And (4) optimizing and determining ion source parameters. The optimization is carried out by adopting Flow Injection Analysis (FIA), the number of optimized ion pairs is limited by an instrument, the research adopts a representative compound for optimization, the representative compound is selected according to the principle that a target analyte with relatively low response is adopted when the compound parameter is optimized, and the optimization result of the ion source parameter is as follows through experiments: air curtain pressure: 30 psi; spraying voltage: 5500V; ion source temperature: 475 ℃; atomizing gas pressure: 40 psi; auxiliary gas pressure: 45 psi.

And setting IDA conditions. In IDA condition setting, the most important is response threshold determination, wherein matrix standard (standard solution prepared by using "blank matrix" extract) is analyzed at screening target concentration level (0.025 μ g/kg), and response threshold is set according to 1/2 degrees of response intensity of lowest response substance. It should be noted that the threshold is matrix dependent, and different matrix thresholds are different, and in general, the threshold setting principle is to ensure that all responses meeting the screening target concentration can trigger EPI collection as much as possible, and simultaneously, the threshold is increased as much as possible to reduce the data collection amount (easy analysis). Two animal-derived substrates (eggs, honey) were examined in this study, with the lowest value set at 3000 cps.

And secondly, the dynamic background deduction function of the instrument is selected in the IDA setting, so that the generation of invalid data can be effectively reduced.

And fourthly, setting EPI parameters.

a) EPI collection mass number range: in the research, the mass number range of the molecular ion peak of 103 target analytes is 140 Da-940 Da, and the mass number range of fragment ions is 80 Da-880 Da, so that the EPI acquisition mass number range is set to be 70 Da-1000 Da;

b) EPI acquisition scan speed: the device used in the research is an AB SCIEX 5500Q-Trap mass spectrometer, EPI acquisition has the speeds of 1000Da/s, 10000Da/s and 20000Da/s of 3, and 10000Da/s is selected as the EPI acquisition scanning speed for ensuring the data quality, so that the analysis acquisition speed of 103 target analytes is considered, and the spectrogram quality is ensured;

c) setting of DP value and EP value of EPI acquisition: in order to ensure the quality of the spectrum library data, the DP values and the EP values of 103 target analytes are subjected to sectional statistics, and the median value of a high-proportion section is taken as a final set value.

Through analysis, the proportion of the DP value of the target compound is increased in a 60V-100V section, and the median value of 80V is taken as the DP set value acquired by the EPI. Similarly, the EP value of the target group compound is larger in the range of 9V to 11V, and the median value of 10V is taken as the EP set value for EPI acquisition. After the DP and EP set values are determined, the DP value and the EP value are acquired, and the set DP value and EP value are proved to have no obvious influence on the EPI data acquisition quality of the compound;

d) referring to table 3, the CE values of the common compounds are generally set to 20eV to 30eV, but when the CE value of the individual compound such as crystal violet is set to 45 to 60eV, the fragment data having high quality is obtained. For this purpose, the EPI spectrum quality was examined by fixing the CE value to 35 and then setting the extended CE value (CES) to 3 combinations of 5, 10, 15, etc. And finally, selecting a CE of 35eV and a CES of 15eV (which is equivalent to the cumulative average of 3 spectrograms when the CEs are respectively 20eV, 35eV and 50 eV) as a CE setting for EPI acquisition, wherein the setting can better give consideration to high, medium and low collision energy sections, and a high-quality data spectrogram can be obtained. Under the chromatographic and mass spectrometric conditions described above, the total ion flow pattern for 115 target analytes (class a + class B) is shown in fig. 1; a typical selected ion flow diagram is shown in fig. 2; the ion flow diagram for extracting each target analyte is shown in fig. 3, and the database spectrogram when CES expansion is selected (namely CE35eV, CES 15eV) is shown in fig. 4.

Example 5 liquid Mass Spectrometry library construction-liquid Mass Spectrometry library construction

By the method of example 4 above, an advanced mass spectrometry acquisition method (sMRM-IDA-EPI) of 115 high-residual risk compounds including 103 target analytes of class B was established, which can be used to perform screening acquisition (using pairs of collected ions of sMRM), and then EPI acquisition is performed on the target that meets the screening rules (concentration response exceeds the screening target concentration), and detailed ion information is obtained for qualitative analysis.

Liquid mass spectrometry library construction

Firstly, acquiring EPI spectrogram data on line. The method comprises the steps of diluting 103 target substance mixed standard working solutions by using a mobile phase to prepare mixed standard solutions with the concentration of a screened target concentration level, collecting online EPI data in an sMRM-IDA-EPI mode through computer analysis, constructing a spectrum library by using Analyst1.5 software, and perfecting target analysis information (such as Chinese and English names, chemical formulas, CAS numbers, chemical structure diagrams and the like).

And secondly, acquiring off-line EPI spectrogram data. The standard stock solution prepared by classification (like the standard stock solution with isomers in the class, the standard stock solution needs to be injected separately) is diluted to 0.2mg/L by using a mobile phase, the sample injection is carried out by direct mass spectrometry, off-line EPI data is acquired according to on-line EPI conditions, in addition, 3 CE level (low, medium and high) EPI spectrograms are acquired separately, and optimized collision energy can be adopted according to the actual properties of the compound. And recording the acquired data into a spectrum library.

And thirdly, judging the use rule of the liquid mass spectrum library. The spectral library retrieval is one of the most effective qualitative means, but no regulation and technical provisions are provided at present in the aspect of matching rule establishment of the liquid mass spectral library retrieval. The research determines the judgment rules of retention time and signal-to-noise ratio according to relevant regulations of European Union and America; determination of EPI spectrogram comparison critical matching factor matrix standards were prepared with 10 "blank matrices" (5 each of honey and egg samples), and subjected to matching analysis with standard EPI spectra in the EPI library using Analyst1.5 software to obtain purity values (purity), and average values and standard deviations. Through comparison, the average value minus the standard deviation of all the target substances is larger than 60, and the study determines that the write value 60 is a critical matching factor for controlling the false negative rate to the maximum extent. In conclusion, the search rule of the spectral library determined by research is as follows:

a) the retention time of the extracted ion current of the compound in the sample is not more than 5% of the retention time of the extracted ion current of the target analyte in the standard solution or the added sample;

b) the parent/daughter ions (transport ion pairs) of the target analytes listed in Table 3 must occur simultaneously and the signal-to-noise ratio (S/N) of the transport ion pairs is ≧ 3;

c) the spectrum matching Purity (Purity value) or the critical matching factor is more than or equal to 60.

The spectrum library has strong qualitative function compared with the spectrum library, and provides more information. According to the specification of European Union 2002/657/EC, the research collection mode can obtain the confirmation point number more than or equal to 5.5 (the most strict banned veterinary drug of the European Union only requires the confirmation point number more than or equal to 4). Thus, rapid qualitative corroboration of the target analyte can be performed if the spectral library search matches.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to determine the protection scope of the present invention.

Claims (5)

1.A construction method of a rapid screening liquid mass spectrum library for detecting various residues of high-risk drugs in animal-derived food specifically comprises the following steps,

(1) preparing a standard working solution;

(2) carrying out analysis pretreatment;

(3) carrying out one-time sample introduction chromatographic analysis;

(4) constructing a liquid mass spectrum library;

the animal-derived food is poultry egg food or honey food,

the high-risk drugs are 103 high-risk residual drugs with a limit value of more than 1.0 mu g/kg, and specifically comprise:

a. the male hormones are 8 types: trenbolone, methyltestosterone, nandrolone propionate, nandrolone phenylpropionate, testosterone propionate, dehydrotestosterone, and dehydromethyltestosterone;

b. 6 progestogens: medroxyprogesterone, medroxyprogesterone acetate, megestrol acetate, acetate progesterone, melengestrol, acetate 17 α -hydroxyprogesterone;

c. glucocorticoids are of 6 types: beclomethasone, fludrocortisone acetate, hydrocortisone, methylprednisolone, prednisone, cortisone;

d. nitroimidazole and metabolite 5 species: isonidazole, metronidazole, hydroxymetronidazole, hydroxyisonidazole;

e. nitrofuran metabolites 4 species: nitrofurazone metabolites, nitrofurantoin metabolites, furazolidone metabolites, furaltadone metabolites;

f. fluoroquinolones are 14 species: sarafloxacin, enrofloxacin, ciprofloxacin, ofloxacin, norfloxacin, lomefloxacin, pefloxacin, sparfloxacin, difloxacin, danofloxacin, marbofloxacin, orbifloxacin, enoxacin, flumequine;

g. 4 quinolones: pipemidic acid, oxolinic acid, pyrrohdic acid, nalidixic acid;

h. beta agonists class 8: salbutamol, terbutaline, ractopamine, salmeterol, fenoterol, methoxytyramine, chlorpropaline, penbutolol;

i. 24 sulfanilamides: sulfadiazine, sulfanilamide-5- (P) methoxy pyrimidine, sulfadimidine, sulfaquinoxaline, sulfadimethoxine, sulfanilamide-6- (m) methoxy pyrimidine, sulfamethoxypyridazine, sulfamethoxazole, sulfathiazole, sulfacetamide, sulfamethoxazole, sulfamethizole, sulfachloropyridazine, sulfadimidine, sulfapyridine, sulfaphenazole, sulfachloropyrazine, sulfamethazine, sulfanitrobenzene, sulfaphenacyl, sulfa-dimethoxypyrimidine, sulfanilamide, sulfaguanidine;

j. 1 sulfanilamide synergist: trimethoprim;

k. macrolides are 11: spiramycin, kitasamycin, tilmicosin, midecamycin, clarithromycin, azithromycin, roxithromycin, josamycin, tylosin, oleandomycin, erythromycin;

lincomycin;

4 of the tetracyclines: tetracycline, oxytetracycline, chlortetracycline, doxycycline;

n. 5 polyethers: salinomycin, methyl salinomycin, monensin, maduramicin, and lasalocid;

o. dyes of 2 types: malachite green, leucomalachite green;

wherein,

the preparation method of the standard working solution in the step (1) comprises the following steps:

a.0.01g/L male hormone substance mixed standard stock solution: comprises trenbolone, methyltestosterone, nandrolone propionate, nandrolone phenylpropionate, testosterone propionate, dehydrotestosterone, and dehydromethyltestosterone, and the preparation solvent is acetonitrile;

b.0.01g/L of progestogen substance mixed standard stock solution: comprises medroxyprogesterone, medroxyprogesterone acetate, megestrol, acetate progesterone, melengestrol and acetate 17 alpha-hydroxyprogesterone, and the preparation solvent is acetonitrile;

c.0.01g/L glucocorticoid substance mixed standard stock solution: comprises beclomethasone, fludrocortisone acetate, hydrocortisone, methylprednisolone, prednisone and cortisone, and the preparation solvent is methanol;

d.0.01g/L of 5-nitroimidazole drugs and metabolites thereof mixed standard stock solution: comprises ipronidazole, metronidazole, hydroxymetronidazole and hydroxyipronidazole, and the preparation solvent is acetonitrile;

e.0.01g/L of Nitrofuran metabolite and 2-nitrobenzaldehyde-derivatized Nitrofuran metabolite mix Standard stock solutions: furacilin metabolite, nitrofurantoin metabolite, furazolidone metabolite, furaltadone metabolite and their respective 2-nitrobenzaldehyde derivatives, the preparation solvent is acetonitrile;

f.0.01g/L fluoroquinolone mixed standard stock solution: comprises sarafloxacin, enrofloxacin, ciprofloxacin, ofloxacin, norfloxacin, lomefloxacin, pefloxacin, sparfloxacin, difloxacin, danofloxacin, marbofloxacin, orbifloxacin, enoxacin and flumequine, and the preparation solvent is acetonitrile;

g.0.01g/L carbostyril substance mixed standard stock solution: comprises pipemidic acid, oxolinic acid, pyrrole acid and nalidixic acid, and the preparation solvent is acetonitrile; h.0.01g/L of beta-receptor agonist substance mixed standard stock solution: comprises salbutamol, terbutaline, ractopamine, salmeterol, fenoterol, methoxytyramine, chlorpropaline and penbutolol, and the preparation solvent is acetonitrile;

mixing standard stock solutions of i & j.0.01g/L sulfonamides and synergists thereof: comprises sulfadiazine, sulfanilamide-5- (p) trimethoprim, sulfadimidine, sulfaquinoxaline, sulfadimethoxine, sulfanilamide-6- (m) trimethoprim, sulfamethoxypyridazine, sulfamethoxazole, sulfathiazole, sulfacetamide, sulfamethoxazole, sulfamethizole, sulfachloropyridazine, sulfadimidine, sulfapyridine, sulfaphenazole, sulfachloropyrazine, sulfamethazine, sulfanitrobenzene, sulfaphenacyl, sulfa-o-dimethoxypyrimidine, sulfanilamide, sulfaguanidine and trimethoprim, and the preparation solvent is acetonitrile;

k.0.01g/L macrolide mix Standard stock solutions: comprises spiramycin, kitasamycin, tilmicosin, midecamycin, clarithromycin, azithromycin, roxithromycin, josamycin, tylosin, oleandomycin and erythromycin, and the preparation solvent is acetonitrile;

l.0.01g/L lincomycin standard stock solution, and the preparation solvent is acetonitrile;

m.0.01g/L tetracycline substance mixed standard stock solution: comprises tetracycline, oxytetracycline, chlortetracycline and doxycycline, and the preparation solvent is acetonitrile;

n.0.01g/L of polyether substance mixed standard stock solution: comprises salinomycin, methyl salinomycin, monensin, maduramicin and lasalocid, and the preparation solvent is methanol;

o.0.01g/L dye class mix Standard stock solution: comprises malachite green and leucomalachite green, and the solvent is methanol;

0.5mg/L of 99 substances mixed standard working solution: transferring 0.5mL of each mixed standard stock solution except the mixed standard stock solution of the nitrofuran metabolite and the 2-nitrobenzaldehyde derivative thereof into a 10mL volumetric flask, metering the volume to a scale by using acetonitrile, and uniformly mixing;

0.5mg/L of 103 substances mixed standard working solution: transferring 0.5mL of each mixed standard stock solution except the mixed standard stock solution of the nitrofuran metabolites into a 10mL volumetric flask, metering the volume to a scale by using acetonitrile, and uniformly mixing;

0.5mg/L nitrofuran metabolite mix standard working solution: respectively transferring 0.5mL of nitrofuran metabolite mixed standard stock solution into a 10mL volumetric flask, metering the volume to the scale with acetonitrile, and uniformly mixing;

the analysis pretreatment method in the step (2) comprises the following steps:

a) weighing 2g of homogeneous sample, placing the homogeneous sample in a 50mL polytetrafluoroethylene centrifuge tube, adding 3.75mL of 0.2mol/L ammonium acetate buffer solution with pH of 5.2 and Na₂EDTA-Mclvaine buffer solution 200 μ L, β -glucuronide/sulfatase 50 μ L, vortex mixing, water bath oscillating at 50 deg.C for 2h, cooling to room temperature, centrifuging at 10 deg.C and 15000rpm for 5min to obtain supernatant as extractive solution A, and keeping;

adding 5mL of 1% formic acid acetonitrile solution into the centrifugal residues, uniformly mixing by vortex, oscillating in a water bath at 50 ℃ for 30min, cooling to room temperature, centrifuging at 10 ℃ and 15000rpm for 5min, blowing the supernatant to 1mL at 30 ℃, adding 3mL of 5mmol/L ammonium formate-0.1% formic acid-water solution to obtain an extracting solution B, and keeping for later use;

respectively placing 400 μ L of the extract A, B in Eppendorf tube, centrifuging at 4 deg.C and 20000rpm for 10min, collecting 400 μ L of supernatant, adding 400 μ L of 5mmol/L ammonium formate-0.1% formic acid-water solution, performing ultrasonic treatment for 1min, filtering with 0.22 μ M microporous membrane, and performing liquid chromatography-tandem mass spectrometry; b) screening furan metabolites by eggs and honey: weighing 2g of uniform sample, placing the sample into a 50mL polytetrafluoroethylene centrifuge tube, adding 3.8mL of 0.1mol/L hydrochloric acid solution and 200 mu L of 0.1 mol/L2-nitrobenzaldehyde solution, uniformly mixing by vortex, oscillating in a water bath at 50 ℃ for 2h, cooling to room temperature, centrifuging at 10 ℃ and 15000rpm for 10min, taking 200 mu L of supernatant, adding 800 mu L of 5mmol/L ammonium formate-0.1% formic acid-water solution, performing ultrasonic treatment for 1min, passing through a 0.22 mu M microporous filter membrane, and performing liquid chromatography-tandem mass spectrometry;

the one-time sample introduction chromatographic analysis method in the step (3) is as follows,

the method comprises the following steps of (1) using a Kinetex C18 chromatographic column, acetonitrile as an organic phase, formic acid as an ionization reinforcing agent, formate as a peak shape optimizing agent, and a combination of water phase/acidic acetonitrile as an optimized mobile phase system;

the concrete conditions are as follows: a chromatographic column: kinetex C18,2.6 μm, 2.1mm × 100mm i.d.;

flow rate: 0.2 mL;

sample introduction amount: 10 mu L of the solution;

column temperature: 30 ℃;

gradient elution procedure is as follows:

time of day Composition of mobile phase 0～2min， 5 percent of A and the balance of B, 2～8min， 20 percent of A and the balance of B, 8～15min， 95 percent of A and the balance of B, 15～16min， 100 percent of A and the balance of B, 16～19min， 100% of A, whichThe rest is B, and the rest is B, 19～20min， 5 percent of A and the balance of B,

the eluent A is: 0.1% formic acid-acetonitrile solution;

the eluent B is as follows: 5mmol/L ammonium formate-0.1% formic acid-water solution.

2. The method of claim 1, wherein the method for constructing the liquid mass spectrum library in the step (4) comprises the following steps:

an advanced acquisition mode of a quadrupole/ion trap tandem mass spectrometry is adopted: presetting multi-reaction detection, information dependence acquisition and enhanced ion scanning;

mass spectrum conditions:

an ion source: an electrospray ion source;

the scanning mode is as follows: scanning positive ions;

the detection mode is as follows: sMRM-IDA-EPI

Electrospray voltage: 5500V;

atomizing gas pressure: 40 psi;

air curtain pressure: 30 psi;

auxiliary gas pressure: 45 psi;

ion source temperature: 475 ℃;

sMRM parameter setting: setting an MRM detection window to be 60s, and setting the scanning time of the target object to be 1.4 s;

IDA rule: response threshold: 3000 cps; dynamic background subtraction; the strongest ion is selected to be 1 to 3;

enhancer ion scan parameter settings: the scanning mass number range is 70-1000 Da; the scanning speed is 10000 Da/s; the scanning accumulation frequency is 1; the collision energy was 35 eV; the extended collision energy is 15 eV;

and (3) aiming at the mass spectrum results of 103 high-risk drugs, constructing a liquid mass spectrum library.

3. The method as claimed in claim 2, wherein the library creating software used in the database construction is analyst1.5 of abciex corporation, and the standard of library creation is set as:

step (1), giving the RT value of each target analyte under the condition system of the set liquid chromatogram in the research;

step (2), providing relevant chemical information of each target analyte;

and (3) giving at least 5 EPI spectra acquired under different conditions for each target analyte, namely 4 EPI spectra with CE of 20eV, 35eV and 50eV, 35eV and CES of 15eV, and at least one EPI spectrum with CE of 35eV and CES of 15eV under chromatographic conditions.

4. The method of claim 3, wherein the design concept setup further comprises a step (4) of trying to show EPI spectra of target analytes in different matrices.

5. A method for the qualitative or quantitative analysis of high-risk drug residues in target animal-derived food products using a rapid screening liquid mass spectrometry library constructed by the method of claim 1, said method comprising the steps of,

(1) a standard solution is prepared and then a standard solution is prepared,

(2) pretreatment of analysis, namely, adopting an extraction and purification technology of rapid enzymolysis and rapid solid-phase extraction to carry out pretreatment of analysis on a sample to be detected,

(3) carrying out chromatography and liquid quality analysis on a sample to be detected to obtain a liquid quality spectrogram, comparing the liquid quality spectrogram with the liquid quality spectrogram library, and carrying out qualitative or quantitative analysis and detection; the qualitative analysis is carried out by searching a spectrum library, and the qualitative standard is as follows:

1) the retention time of the extracted ion current of the compound in the sample is not more than 5% of the retention time of the extracted ion current of the target analyte in the standard solution or the added sample;

2) the parent/daughter ions of the target analyte must appear simultaneously, and the signal-to-noise ratio of the transmitted ion pair is greater than or equal to 3;

3) comparing the EPI spectrogram of the compound in the sample with the EPI spectrogram of the standard solution or the matrix standard solution with the similar concentration level in the spectrum library, wherein the spectrogram matching purity is more than or equal to 60;

the method of quantitative analysis is as follows:

quantifying by using an external standard single-point method, calculating the content of the target analyte according to the formula (1),

formula (1):

wherein:

x-the amount of target analyte in the sample, μ g/kg;

c-concentration of target analyte in sample solution,. mu.g/L;

v, the volume of the sample solution is determined to be mL;

m-mass of sample, g;

r-recovery,%.