CN113295790A - Method for detecting veterinary drug compound residue in livestock meat and byproducts thereof - Google Patents

Abstract

The invention belongs to the field of veterinary drug residue detection, and particularly relates to a method for detecting veterinary drug compound residue in livestock and poultry meat and byproducts thereof. The method of the invention comprises the following steps: (1) establishing a multi-dimensional electronic identity database of a veterinary drug compound standard, comprising: (1) preparing a sample to be detected into a test solution; (2) detecting the test solution by adopting UHPLC-Q/Orbitrap to obtain a chromatogram and a mass spectrum; (3) obtaining a detection result of the veterinary drug compound residue according to the chromatogram and the mass spectrum; wherein, the chromatographic conditions in the step (2) are as follows: the chromatographic column adopts C₁₈Column, mobile phase a is an aqueous solution containing 0.1% formic acid and 20mmol/L ammonium acetate, and mobile phase B is acetonitrile. The technical scheme provided by the invention can carry out non-targeted batch, quick and efficient detection on veterinary drug compound residues in various animal-derived foods. The detected veterinary drug compounds are more comprehensive in variety, more meet the requirements of practical application and have high contentThe application value of (2).

Description

Method for detecting veterinary drug compound residue in livestock meat and byproducts thereof

Technical Field

The invention belongs to the field of veterinary drug residue detection, and particularly relates to a method for detecting veterinary drug compound residue in livestock and poultry meat and byproducts thereof.

Background

The livestock meat such as pig, cattle, sheep, chicken and the like and the livestock byproducts such as pig liver, chicken liver and the like are main sources for human beings to take high-quality protein, fat, essential amino acid and other nutrients, are one of main food raw materials for three meals a day, and the consumption demand of people on animal-derived food is increasing along with the continuous improvement of the living standard of residents in China. The special effects of the veterinary drug comprise disease prevention and treatment, growth improvement promotion and the like, and the veterinary drug plays an important role in the aspects of protecting the growth of food animals, improving industrial income, guaranteeing the storage quality of animal-derived food and the like. However, the veterinary drug is a 'double-edged sword' with one handle, the contradiction between population growth and food supply can be greatly relieved by reasonable use, and the excessive use not only directly affects the food safety, but also indirectly affects the ecological environment and animal-derived food trade. The veterinary drug residue safety problem is a main problem in animal-derived food safety, and the veterinary drug safety problem in food has been highly valued by government departments and widely concerned by people. Therefore, the detection of veterinary drug residues is a very important task.

However, the animal meat and by-product substrates are complex, and the content of various veterinary drugs is very small and the polarity difference is large, so that it is difficult to obtain the information of various veterinary drug residues by one-time detection. The traditional veterinary drug residue detection methods comprise an enzyme-linked immunosorbent assay, a liquid chromatography-mass spectrometry method and the like, but most of the methods can only detect a small amount of veterinary drugs with the same basic structure, and the simultaneous detection of a large amount of veterinary drugs with different chemical structures is difficult to realize.

The Chinese invention patent CN111060638A, a screening and confirming method of 207 veterinary drugs and additives in animal food, proposes a method for simultaneously detecting 207 veterinary drug compounds by using UHPLC-Q/Orbitrap, however, the types of the detected veterinary drug compounds can not cover the mainstream veterinary drug types such as partial hormones, macrolide antibiotics, zearalanol compounds and the like, and can not meet the requirements of practical application. Thus, there remains a need to provide additional assays that cover a more complete spectrum of veterinary compounds.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for detecting veterinary drug compound residues in livestock meat and byproducts thereof, which realizes detection of veterinary drug compounds of more comprehensive types based on UHPLC-Q/Orbitrap.

A method for detecting veterinary drug compound residue in livestock meat and byproducts thereof comprises the following steps:

(1) preparing a sample to be detected into a test solution;

(2) detecting the test solution by adopting UHPLC-Q/Orbitrap to obtain a chromatogram and a mass spectrum;

(3) obtaining a detection result of the veterinary drug compound residue according to the chromatogram and the mass spectrum;

wherein, the chromatographic conditions in the step (2) are as follows: the chromatographic column adopts C₁₈Column, mobile phase A is 0.02-0.1%Formic acid and 2.5-20mmol/L ammonium acetate, and the mobile phase B is acetonitrile.

Preferably, the veterinary drug compound is at least one selected from the group consisting of androgens, oestrogens, progestogens, glucocorticoids, zearalanol compounds, beta-receptor agonists, sulphonamide antibiotic drugs, quinolone antibiotics and macrolide antibiotics.

Preferably, the veterinary drug compound is selected from the group consisting of dehydrotestosterone, epitestosterone, fluoxymesterone, methyltestosterone, nandrolone propionate, nandrolone, testosterone propionate, trenbolone, meperigerone, nandrolone phenylpropionate, stanozolol, norandrostenedione, androstenedione, 17 alpha-methylisotosterone, meindrosterone, epiandrosterone, 17 beta-hydroxyandrostan-3-one, mesterone, danazol, altrenogest, chlordesogestrel acetate, levonorgestrel, medroxyprogesterone acetate, megestrol acetate, norelgestromin acetate, norethisterone acetate, 21-alpha-hydroxyprogesterone, norethindrone, 17-alpha-hydroxyprogesterone, beclomethasone propionate, betamethasone, cortisone acetate, hydrocortisone, methylprednisolone, prednisolone, triamcinolone, cortisone, dexamethasone, hydrocortisone acetate, hydrocortisone acetate, norgestimate, droxysone, and a, Beclomethasone, flumethasone, budesonide, fluocinonide, fludrocortisone acetate, fluorometholone, clobetasol propionate, aldosterone, prednisone, estriol, estrone, dienestrol, diethylstilbestrol, estradiol, octylphenol, 4-nonylphenol, bisphenol A, ethinylestradiol, alpha-zearalenol, beta-zearalenol, zearalenone, alpha-zearalanol, beta-zearalanol, clenbuterol, ractopamine, chlorpromazine, penbuterol, metaproterenol, formoterol, fenoterol, secobronol, bambuterol, salbutamol, terbutaline, tuobuterol, cimaterol, mabuterol, pyrazolsulfaphenamide, sulfapyridine, sulfacetamide, sulfamethoxydiazine, sulfamethoxazole, sulfamethazine dimethyidipine, sulfamethazine, At least one of sulfamethoxazole, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxine, sulfamodiethoxypyrimidine, sulfamchloropyrazine, sulfamamidine, sulfadiazine, sulfathiazole, sulfamethazine, sulfa nitrobenzene, orbifloxacin, danofloxacin, enrofloxacin, flumequine, fleroxacin, ciprofloxacin, lomefloxacin, nalidixic acid, norfloxacin, fleroxacin, sarafloxacin, difloxacin, sparfloxacin, enoxacin, ofloxacin, erythromycin, kitasamycin, lincomycin, tylosin, tilmicosin, tavermectin, oleandomycin, doramectin, ivermectin, and roxithromycin.

Preferably, in the step (1), the method for preparing the test solution comprises: homogenizing the sample to be detected, extracting with acetonitrile aqueous solution containing formic acid, taking supernatant, performing reverse phase solid phase extraction, blow-drying, and fixing the volume to obtain the product.

Preferably, in the step (1), the aqueous acetonitrile solution containing formic acid is an aqueous acetonitrile solution containing 0.2% formic acid with a volume fraction of 80%;

and/or the dosage ratio of the sample to be detected to the acetonitrile aqueous solution containing formic acid is 2g:10 mL;

and/or adding uniform protons, performing vortex oscillation at 2000r/min for 30min, centrifuging at 9500r/min for 5min, and collecting the supernatant;

and/or the solid phase extraction column packing of the reverse phase solid phase extraction is Oasis PRIME HLB;

and/or the amount of the solid phase extraction column packing of the reverse phase solid phase extraction is 3cc 200 mg;

and/or the speed of passing through a solid phase extraction column in the reverse phase solid phase extraction is 1 drop/s;

and/or after constant volume, performing filtration membrane operation on the sample solution, wherein the filtration membrane is a polytetrafluoroethylene membrane.

Preferably, in step (2), the chromatographic conditions further comprise: the chromatographic column is Waters Acquity BEH C₁₈(ii) a And/or, the temperature of the chromatographic column is 30-40 ℃; and/or, flow rate: 0.3-0.5 mL/min; and/or, the sample injection amount is 5-15 mu L;

and/or, a gradient elution procedure:

and/or, the mass spectrometry conditions are:

heating an electrospray ion source; scanning and collecting mode: collecting positive and negative ions simultaneously, and performing primary full scanning/data-dependent secondary scanning; and/or, the spray voltage: 3.5kV (+), -3.0kV (-); and/or, ion transfer tube and heater temperatures: 325 ℃ and 450 ℃; and/or, sheath gas and auxiliary gas flow rates: 35arb, 10 arb; and/or, acquisition range: 100-1000 m/z; and/or, resolution: full MS 75000, dd-MS²17500 (mm); and/or, stemped (n) CE (20%, 40%, 60%); and/or, maximum capacity of ion number in C-Trap: full MS 3X 10⁶、dd-MS²2×10⁵(ii) a And/or, the maximum injection time in the C-Trap: full MS 100MS, dd-MS²50ms；

And/or, the tuning correction of the mass axis of the Q/Orbitrap mass spectrum is as follows: injecting the Calmix correction fluid into a mass spectrometer, debugging and observing the characteristic ion peak of the Calmix correction fluid, and automatically correcting by tuning application software when the ion peaks of positive ions and negative ions are stable.

Preferably, the method further comprises the step of establishing a multidimensional electronic identity database of veterinary drug compound standards, wherein the multidimensional electronic identity database comprises: basic information, chromatographic information and mass spectrum information of the veterinary drug compound standard;

and (3) comparing the chromatogram and the mass spectrogram with the information in the multi-dimensional electronic identity database to obtain a detection result of the veterinary drug compound residue.

Preferably, the basic information comprises Chinese and English names, CAS numbers, chemical molecular formulas and theoretical precise molecular mass numbers of the compounds;

and/or, the chromatographic information comprises retention time;

and/or the mass spectrum information comprises a parent ion mass number, a fragment ion mass number, an ion addition mode and a standard curve of mass spectrum response area to standard substance concentration.

Preferably, the multidimensional electronic identity database comprises a first-level accurate mass number fingerprint identification database and a second-level HCD fragment ion reference spectrogram confirmation database;

the first-level accurate mass number fingerprint identification database is formed by sequentially recording basic information, chromatographic information and mass spectrum information of the veterinary drug compound in Trace Finder data acquisition and processing software;

the secondary HCD fragment ion reference spectrogram confirmation library is formed by adding and recording secondary HCD fragment ion mass spectrograms obtained by cracking veterinary drug compounds under the optimal collision energy in a high-energy collision pool in mzVault spectrogram management software, wherein the optimal collision energy is the recorded collision energy value CE (20%, 40%, 60% ev) when 3-5 fragment ions with the largest selective ion abundance ratio appear.

Preferably, in step (3), the criterion for determining the positivity of a veterinary drug compound is that the raw data extracted within the 5ppm mass window satisfies all of the following conditions:

1) the retention time error range of the mass spectrum peak of the parent ion is within +/-15 s;

2) the mass number deviation of the parent ions is less than or equal to 5 ppm;

3) the matching number of fragment ions in the secondary mass spectrogram is more than or equal to 2, and the mass number deviation of the fragment ions is less than or equal to 10 ppm.

The technical scheme of the invention has the following beneficial effects:

(1) the method optimizes the extraction conditions, chromatographic conditions and mass spectrum conditions of the livestock meat and byproducts thereof, so that the detection method can realize the detection of the residues of 9 veterinary drug compounds such as male hormone, female hormone, progestogen, glucocorticoid, zearalanol compounds, beta-receptor agonist, sulfonamide antibiotics, quinolone antibiotics, macrolide antibiotics and the like by one-time test. Compared with other prior art, the veterinary drug compound detected by the invention is more comprehensive in types and more meets the requirements of practical application.

(2) In a preferable scheme, the invention provides a construction method and a detection platform of a multi-dimensional electronic identity database of a veterinary drug compound in animal-derived food based on UHPLC-Q/Orbitrap, the multi-dimensional electronic identity database of the veterinary drug compound is established based on the UHPLC-Q/Orbitrap technology on the basis of obtaining chromatographic and mass spectrum information, and a real object standard substance is replaced by an electronic identity, so that automatic, multi-target, high-precision and high-efficiency non-targeted veterinary drug residue detection can be realized. The method has important significance for realizing multi-target quick locking and discrimination confirmation of hundreds of trace veterinary drug residues in animal-derived food. The method of the invention is simpler and more efficient when being used for batch processing of various veterinary drug compounds, has lower requirements on knowledge and experience level of operators, and is beneficial to wide application of the technology of the invention.

(3) In a preferred scheme, the high-throughput detection method based on the multi-dimensional electronic identity database and the detection platform of the veterinary drug compound replaces the traditional targeted qualitative analysis method based on the reference of a real standard, and the leap-type development of the veterinary drug residue from the targeted detection of the known analyte to the high-throughput non-targeted detection technology of the unknown analyte is realized. The method has the advantages of shortening sample analysis time, improving sample detection efficiency, saving resources, reducing pollution and meeting the requirements of sustainable development and environmental friendliness.

(4) In the preferred scheme, the database (MS) is identified by first-level accurate mass number fingerprint identification through searching and comparing the acquired information of the veterinary drug compound to be detected and the multidimensional electronic identity information in the database according to the retention time, accurate mass number, ion abundance ratio, collision energy, second-level HCD fragment ion graph and other information of the target compound¹) Screening and identifying the medium-color mass spectrum fingerprint information, and matching with a secondary HCD fragment ion reference spectrogram verification library (MS)²) The medium characteristic spectrogram confirms, and the accuracy of the accurate and qualitative screening result of the medicine is ensured.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 ion current chromatogram for extraction of positive components from beef sample in example 1.

FIG. 2 Experimental example 1 128 veterinary drugs in Waters Acquity BEH C₁₈Selective ion chromatogram of (1).

FIG. 3 ion chromatogram of peak profile optimization of 3 typical veterinary drugs in Experimental example 1

FIG. 4 Secondary mass spectra of three typical "clenbuterol" compounds in Experimental example 1.

Detailed Description

The instruments, reagents and materials used in the examples and experimental examples are as follows:

(1) laboratory apparatus

UHPLC/Q active Plus ultra high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometry system (with HESI ion source, Trace Finder data acquisition and processing software and mzVault spectrogram management software, U.S. Thermo Fisher company); Milli-Q ultrapure water system (Millipore, USA); electronic balance model ME203 (Mettler Toledo, Switzerland), Waters Acquity BEH C₁₈Column (2.1 mm. times.100 mm, 1.7 μm, Waters Corp.).

(2) Experimental reagent

Acetonitrile and methanol are both mass spectrum grades (Thermo Scientific corporation, usa); formic acid, ammonium acetate are both mass spectral grades (Sigma Aldrich, usa); ceramic homogenous protons (Agilent corporation, usa); oasis HLB, Oasis PRiME HLB solid phase extraction columns (Waters corporation, usa); captiva EMR Lipid solid phase extraction column (Agilent, USA); 25 ℃ to 18.2M Ω cm ultrapure water (Millipore, USA); the purity of 128 veterinary drug standards is more than or equal to 90.0 percent (Dr. Ehrenstonerfer GmbH, Germany, Sigma Aldrich, Cato Research Chemicals Inc, China food and drug testing Research institute, Tianjin Alta science and technology Co., Ltd.)

(3) Experimental Material

Livestock and poultry meat of 112 batches and byproducts (pork, beef, mutton, chicken of 24 batches respectively, pork liver and chicken liver of 8 batches respectively) are purchased from local supermarkets and farmer markets.

Example 1

Firstly, preparation of standard solution

(1) Standard stock solutions: accurately weighing 2mg of each standard substance, sequentially diluting with acetonitrile, diluting to a constant volume of 1mg/mL of standard stock solution, and storing at-18 ℃ in a dark place.

(2) Mixing standard working solution: accurately transferring 100 mu L of each standard stock solution into a 10mL volumetric flask, diluting the standard stock solutions to the scales by using acetonitrile to prepare 16 mixed standard intermediate solutions I with the concentration of 10 mu g/mL, accurately transferring 100 mu L of each mixed standard intermediate solution I, metering the volume to 10mL by using the acetonitrile to prepare a mixed standard intermediate solution II with the concentration of 100 ng/mL; then accurately transferring 0.1 mL, 0.5mL, 1mL, 2mL and 5mL of the mixed standard intermediate solution II to prepare mixed standard working solutions with the concentrations of 1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL and storing at minus 18 ℃ in a dark place.

Second, high flux sample pretreatment method

Preparing samples of livestock and poultry meat and byproducts thereof according to the requirements of GB/T30891-. Taking 5mL of supernatant, purifying by a solid phase extraction column, collecting the effluent liquid of the latter half section of 4mL, accurately measuring 2mL, drying by nitrogen until the volume is 1mL, fixing the volume of the initial mobile phase, filtering by a membrane, and waiting for testing on a machine. 5mL of the supernatant was collected and passed through 6cc of 200mg Oasis PRIME HLB solid phase extraction column at 1 drop/s. Collecting the latter half-section effluent of 4mL, accurately measuring 2mL, drying under nitrogen, metering volume of 1mL of initial mobile phase, and filtering through a polytetrafluoroethylene filter membrane of 0.22 mu m to obtain a test solution for UHPLC-Q/Orbitrap test.

Third, one-time sample introduction is carried out for UHPLC-Q/Orbitrap instrument analysis

The instrument analysis conditions were as follows:

UHPLC conditions: a chromatographic column: waters Acquity BEH C₁₈(2.1 mm. times.100 mm, 1.7 μm); temperature of the column: at 40 ℃. Flow rate: 0.5 mL/min; mobile phase a (aqueous phase) was 20mmol/L ammonium acetate in water with 0.1% formic acid and mobile phase B (organic phase) was acetonitrile. Gradient elution procedure: the initial mobile phase a phase was first 98% to peak the polar species first, then the initial acetonitrile concentration was reduced and maintained to slowly increase to 100%, and the acetonitrile concentration was reduced to the initial concentration level and maintained at equilibrium. The gradient elution program settings are shown in table 1. Sample introduction amount: 10 μ L.

Table 1 liquid chromatography mobile phase gradient elution procedure

Q/Orbitrap high resolution mass spectrometry conditions: an ion source: heating an electrospray ion source; scanning and collecting mode: simultaneous positive and negative ion acquisition, first-order Full scan/data-dependent second-order scan (Full MS/dd-MS)²) (Top N); spraying voltage: 3.5kV (+), -3.0kV (-); ion transfer tube and heater temperatures: 325 ℃ and 450 ℃; sheath gas and auxiliary gas (N)₂) Flow rate: 35arb, 10 arb; the collection range is as follows: 100-1000 m/z; resolution ratio: 75000(Full MS), 17500 (dd-MS)²) (ii) a Stepped (N) CE (20%, 40%, 60%); maximum capacity of ion number in C-Trap (AGC target): 3X 10⁶(Full MS)、2×10⁵(dd-MS²) (ii) a Maximum injection time in C-Trap: 100MS (full MS), 50MS (dd-MS)²)。

Fourthly, establishing a 128 veterinary drug multi-dimensional electronic identity database

The multidimensional electronic identity database is used as an electronic identity card database of veterinary drugs, and can be used for carrying out quick locking recognition and discrimination confirmation on 128 veterinary drugs under the condition of no standard substance contrast in the later period. The multi-dimensional electronic identity database of veterinary compounds comprises basic information, chromatographic information and mass spectral information of veterinary compound standards. The basic information comprises Chinese and English names of the compounds, CAS numbers, chemical molecular formulas and theoretical accurate molecular mass numbers; the chromatographic information comprises retention time; the mass spectrum information comprises a parent ion mass number, a fragment ion mass number, an ion addition mode and a standard curve of mass spectrum response area to standard substance concentration.

The multi-dimensional electronic identity database comprises a first-level accurate mass number fingerprint identification database (MS)¹) And secondary HCD fragment ion reference spectrum validation library (MS)²). The first-class accurate mass number fingerprint identification database is formed by sequentially recording basic information, chromatographic information and mass spectrum information of the veterinary drug compound in Trace Finder data acquisition and processing software. The secondary HCD fragment ion reference spectrum confirmation library is formed by adding and recording a secondary HCD fragment ion mass spectrum obtained by cracking a veterinary drug compound in a high-energy collision pool under the optimal collision energy in mzVault spectrum management software, wherein the optimal collision energy is a collision energy value recorded when 3-5 fragment ions with the maximum selective ion abundance ratio appear, and Stepped (N) CE (20%, 40%, 60%).

In this example, a multidimensional electronic identity database of 128 veterinary compounds was created according to the above method, and the specific information of the 128 veterinary compounds is shown in the following table:

TABLE 2 UHPLC-Q/Orbitrap 128 veterinary drug Compound information

Fifth, data processing and analysis

In TraceFinder analysis software, the established high resolution mass spectrum Database is used to search, match and analyze the sample data collected by UHPLC-Q/Orbitrap, and the automatic deconvolution function of the mass spectrum software is used to compare with the retention time, accurate parent ion mass number, fragment ion mass number, secondary mass spectrogram and other relevant parameters in the Compound Database (Compound Database) for discrimination.

The criterion for determining the positivity of a veterinary drug compound is that the raw data extracted within a 5ppm mass window satisfies all of the following conditions:

1) the retention time error range of the mass spectrum peak of the parent ion is within +/-15 s;

2) the mass number deviation of the parent ions is less than or equal to 5 ppm;

3) the matching number of fragment ions in the secondary mass spectrogram is more than or equal to 2, and the mass number deviation of the fragment ions is less than or equal to 10 ppm.

For the veterinary drug compound judged to be positive, the mass spectrum response area of the veterinary drug compound can be substituted into a standard curve in a multidimensional electronic identity database, and the content of the veterinary drug compound in livestock meat and byproducts thereof can be calculated.

By the method, 112 batches of samples (wherein each 24 batches of pork, beef, mutton and chicken and each 8 batches of pork liver and chicken liver) are detected. 24 veterinary drug residues are detected, the veterinary drug residues are detected for 71 times in total, and the samples are 71 batches (17 batches of pork and chicken, 14 batches of beef and 11 batches of mutton). The specific results are shown in Table 3.

TABLE 3 detection results of veterinary drug residues in livestock and poultry meat and by-products

The control veterinary drug limit national standard GB 31650 and 2019 require that most of 128 veterinary drugs exceed the method quantitative limit but are lower than the limit value of the national standard. Wherein, the limited veterinary drug dexamethasone and the limited veterinary drug lincomycin are simultaneously detected in 1 batch of beef. Problem extraction of positive compounds from beef samples is shown in figure 1.

Experimental example 1 optimization of analysis conditions of UHPLC-Q/Orbitrap Instrument

(1) Selection of chromatographic column and column temperature

The invention needs to realize effective separation of veterinary drugs with different 128 properties, and needs to select a chromatographic column with good compatibility, good column efficiency and durability and column temperature. Experiment firstly compares Thermo Accucore aQ C₁₈(2.1mm×100mm，2.6μm)、Agilent Eclipse plus C₁₈(3.0 mm. times.150 mm, 1.8 μm) and Waters Acquity BEH C₁₈(2.1 mm. times.100 mm, 1.7 μm) separation effect of three kinds of columns on 128 kinds of compounds. Part of veterinary drug is in Thermo Accucorea Q C₁₈And Agilent Eclipse plus C₁₈The chromatographic columns have different degrees of peak-type tailing and poor peak-type tailing. While Waters Acquity BEH C₁₈The chromatographic column can better retain 128 hormone compounds, and the peak type has no obvious tail and is sharp and symmetrical.

The chromatographic column temperature often influences the separation efficiency of the coloration spectrum, and experiments investigate the peak effect under 4 different column temperatures (increasing by 5 ℃ within the range of 20-50 ℃), and the experiments find that: in the range of 20-25 ℃, the peak effect is obviously and greatly enhanced along with the rise of the temperature. ② in the range of 40-50 ℃, the retention time and the peak effect of most compounds are not obviously changed. In order to save energy and time, 40 ℃ was chosen as the column temperature. 128 veterinary drugs in Waters Acquity BEH C₁₈The selective ion chromatogram above is shown in FIG. 2.

(2) Selection of mobile phase system and flow rate

The mobile phase system and flow rate settings directly affect the retention time and peak pattern of the analyte. The 128 drugs involved in the experiment can be easily dissolved in acetonitrile, and are middle and weak polarity drugs. 4 different (acetonitrile-water and ammonium acetate with different concentrations added under the condition) flow combinations are respectively selected for mass spectrometry, and the results show that: 0.1% formic acid is added into acetonitrile-20 mmol/L ammonium acetate solution, at this time, the chromatographic peak shape of each substance to be measured is best, and the matrix interference is minimum. Thus, the aqueous phase was finally selected to be 0.1% formic acid-ammonium acetate (20mmol/L) in water and the organic phase to be acetonitrile.

Meanwhile, the separation effect of the three different flow rates on the object to be detected is investigated. The result shows that the separation of 128 veterinary drugs can be realized at three flow rates (0.3/0.4/0.5, mL/min), when the flow rate is 0.5mL/min, the chromatographic peak is sharp, the recovery rate is high, the detection efficiency can be effectively improved, and the requirements of veterinary drug multi-residue analysis and detection are met. The ion chromatogram after peak pattern optimization of 3 typical veterinary drugs is shown in figure 3.

(3) Selection of gradient elution procedure

The invention aims to realize the multi-target simultaneous detection of 128 veterinary drugs in 6 matrixes, and adopts gradient elution to realize complete separation. The gradient elution procedure is set up in Table 1, taking into account the peak type, sensitivity, time to peak, and the like.

(4) Selection of scan acquisition mode

The invention is directed to Full MS and Full MS/dd-MS²And selective ion scanning (Target-SIM)/dd-MS²Three scanning acquisition modes are investigated and contrasted, and test results show that: although quantitative detection can be completed in a Full MS mode, qualitative aspects only depend on the accurate mass number and retention time of parent ions as reference bases, and qualitative inaccuracy is easily caused to the deficiency of fragment ion analysis; Target-SIM/dd-MS²The mode takes the accurate mass numbers of the parent ions and the fragment ions as qualitative basis to improve the qualitative capability of the target object, but the quantitative level is not good enough. Full MS/dd-MS²Qualitative (two-stage ion accurate mass number) screening and quantitative (one-stage ion) detection of the target veterinary drug can be simultaneously realized, and the accurate relative mass deviation of the 128 compounds is less than 1ppm under the condition, so that the experimental requirements are met. Therefore, the scanning mode is Full MS/dd-MS²。

(5) Selection of mass spectral resolution

The mass resolution of a mass spectrometry scan determines the accuracy of the ion mass number, and usually requires both selectivity and sensitivity. The invention sets various data for the resolution of the primary and secondary mass spectra and carries out comparative analysis, and experiments show that when the resolution R of the primary full-scanning mass spectrum is 140000 (ultrahigh resolution), the scanning selectivity is very strong but the sensitivity is reduced, and the number of collected points is too small; when the first-stage full-scan mass resolution R is 75000 (high resolution), the ratio of baseline separation between the analyte and the impurity is optimal and the response value is improved. The two-stage mass spectrometry scan resolution 75000/17500.

(6) Selection of secondary mass spectrum parameters

The target objects are listed on the basis of full scanning, and tests under different threshold conditions show that the intensity threshold of the secondary mass spectrum when automatically triggered is 50000. In addition, it is found that the number of secondary mass spectrum acquisitions of some substances is small, especially, the secondary mass spectrum of which the number of individual isomer substances is only within two can be acquired, and the cause of such phenomenon may be related to factors such as poor parameter setting range of Dynamic exclusion (Dynamic exclusion) or open Apex trigger (Apex trigger). Firstly, the half-peak width is confirmed to be the optimal time of the Dynamic exception, the time obtained after deep optimization is respectively 6s and 3s, and the Dynamic exception is finally determined to be 3s in consideration of the requirement of considering different peak widths of 128 veterinary drugs. And secondly, fragmenting the compound by adopting three different normalized collision energies (Stepped (N) CE) of 20%, 40% and 60% to obtain an additive graph with rich fragment ion information. Thirdly, under the condition of opening vertex excitation, the fact that a single compound is difficult to excite the second-order at the vertex is found, which is probably caused by the fact that the number of the compounds to be detected is large and obvious peak width difference exists among all substances, so that the vertex excitation function is not started, and a satisfactory second-order mass spectrum can be obtained when the first three-intensity (TopN is 3) is acquired. The secondary mass spectra of three typical "clenbuterol" compounds under selected secondary mass spectrometric conditions are shown in FIG. 4.

Experimental example 2 optimization of sample pretreatment method

Various matrix interfering impurities exist in livestock and poultry meat and byproducts, and the interfering substances not only pollute a chromatographic column, but also can cause serious matrix inhibition, and finally influence the sensitivity and accuracy of the determination of the drug to be detected. In the experimental example, the pretreatment technology of the passing-type reversed-phase solid-phase extraction sample is optimized by selecting plates such as extraction, purification and needle type filter membranes.

(1) Selection of extraction solvent, volume of extraction solvent and extraction times

The extraction reagent can extract veterinary drugs from a sample matrix, and different extraction reagents have obvious effect difference. Common extractants for veterinary drugs comprise ethyl acetate, acetonitrile aqueous solution and the like, and the experimental example examines the extraction effects of 6 extractants such as ethyl acetate, acetonitrile, 80% acetonitrile aqueous solution, 90% acetonitrile aqueous solution, 0.2% formic acid-80% acetonitrile aqueous solution, 1.0% formic acid-80% acetonitrile aqueous solution and the like on 128 veterinary drugs.

The results show that: the extraction efficiency of the acetonitrile water solution is obviously higher than that of ethyl acetate and pure acetonitrile, the extraction efficiency of the acetonitrile water solution with 90% volume fraction is lower than that of the acetonitrile water solution with 80% volume fraction, the addition of a proper amount of formic acid can effectively increase the stability of veterinary drugs, precipitate protein in a sample at a high level and realize the extraction of a target object, and the extraction efficiency of 0.2% formic acid is higher than that of 1.0% formic acid. Therefore, the 0.2% formic acid-80% acetonitrile water solution has better extraction efficiency compared with other 5 extraction solvents.

To extract the target substance as completely as possible, an appropriate volume of the extraction solvent needs to be determined. In this experimental example, 80% acetonitrile aqueous solution (containing 0.2% formic acid) was used as the extraction solvent, and the influence of the volume of the extraction solvent on the extraction effect of the drug to be tested was examined for three types, 5, 10 and 15mL, respectively. The results show that the veterinary drug extraction effect in the samples was ranked as high or low at three different extraction solvent volumes: 10mL > 15mL > 5 mL. When the extraction solvent is 10mL, the target compound hexylidene is completely extracted, and the extraction effect is best.

The meat and by-products of livestock and poultry are mostly solid matrix, which is not miscible with the extraction solution. The experimental example is to extract the veterinary drug for 3 times on the basis of selecting the extraction solvent, and the influence of the extraction times on the veterinary drug extraction effect is evaluated. The results show that there was no significant difference in the extraction effect of the drug in the sample in the case of 1 extraction, 2 extractions, and 3 extractions, probably because 1 extraction of the drug in 0.2% formic acid-80% acetonitrile aqueous solution had achieved complete extraction of the drug. And comprehensively considering the aspects of working efficiency, experiment cost and the like, and selecting 1 extraction.

(2) Optimization of sample homogeneous centrifugal extraction conditions

In the veterinary drug residue detection process, through homogenization and centrifugation, the components can be dispersed after the samples are fully and uniformly mixed, and good consistency is achieved. In this example, the sample is uniformly mixed and dispersed under the above experimental conditions under 3 homogeneous centrifugation conditions (a. homogeneous seed + vortex oscillation 2000r/min + 30min + centrifugation 9500r/min + 5 min; b. homogeneous proton + vortex oscillation 2000r/min + 30min +6000r/min + 10 min; c. vortex oscillation 2000r/min + 30min +9500r/min + 5min), and the results show that the dispersion and extraction effects of the sample are: a > B > C.

(3) Selection of solid phase extraction column and amount of packing

The matrix of livestock and poultry meat and byproducts, especially animal liver samples is complex, and the samples still need to be purified after being extracted. This example examines C₁₈The four extraction columns of Oasis HLB, Oasis PRIME HLB and Captiva EMR Lipid have separation and purification effects on veterinary drugs. When using C₁₈The average recovery rate of 128 veterinary drugs is the lowest, which indicates that the traditional C is the lowest₁₈The adsorbent is difficult to simultaneously consider all veterinary drug properties, the purification effect of the Oasis PRIME HLB is obviously superior to that of the Oasis HLB and Captiva EMR Lipid, and the Oasis PRIME HLB can better adsorb phospholipid, protein and other impurities in a sample.

The experimental example simultaneously examines the adsorption and purification effects of different filler dosages (1cc 30mg/3cc 60mg/3cc 150mg/6cc 200mg/6cc 500mg) of Oasis PRIME HLB on the target. The results show that the extraction efficiency increases rapidly when the packing is increased from 1cc 30mg to 3cc 200 mg; when the filling amount is 6cc 200mg and 6cc 500mg, the absorption and purification effects of the filling amount and the filling amount on the livestock and poultry meat are equivalent, but the absorption effect of the filling amount and the absorption and purification effects on the livestock and poultry byproduct matrix is the best.

(4) Selection of needle type Filter membranes

The needle type filter membrane mainly plays a role in filtering particulate matters in the test solution on the computer, and a small amount of floccules still suspend in the test solution after the sample is extracted and purified. This example examined the effect of purifying veterinary drugs with three filters, 0.22 μm polytetrafluoroethylene (A), 0.22 μm nylon filter (B) and 0.22 μm polyethersulfone (C). The results show that after the sample solution is filtered by needle type filter membranes made of three different materials, a large amount of compounds are detected, and the filtering effects of the three filter membranes are as follows: 0.22 mu m of polytetrafluoroethylene is more than 0.22 mu m of nylon filter membrane and more than 0.22 mu m of polyether sulfone.

Experimental example 3 methodological investigation and verification

(1) Evaluation of matrix Effect

Compared with a low-resolution mass spectrum such as a triple quadrupole and the like, the UHPLC-Q/Orbitrap high-resolution mass spectrum has obvious advantages in the aspect of anti-interference capability, but still does not fundamentally solve the problem of matrix effect. The samples involved in this study are of many varieties and complex matrices, so matrix effect evaluation is required. The internal standard method has the best effect of reducing the influence of matrix effect, but the research relates to too many types of medicines, and the internal standard is expensive and unavailable, so the response surface method is adopted as a research method in the embodiment. The 128 veterinary drug standards are respectively prepared into standard solutions by 6 types of matrixes such as pork, beef and the like and methanol, and the matrix effect condition of the compound is evaluated by the ratio of the slope of a standard curve of the same substance in the pure methanol solvent and the matrix solution. The results show that the 6 types of substrates have substrate effect, and particularly the substrate effect of pork and beef substrates is more obvious. The sample has a matrix effect, which can affect the quantitative determination of part of compounds to a certain extent, the matrix effect can be effectively reduced through sample pretreatment and instrument condition optimization in the early stage, and the influence of the matrix effect can be eliminated or weakened through matrix matching with a standard curve in the quantitative link.

(2) Linear range, detection limit, and quantitation limit of a method

Based on UHPLC-Q/Orbitrap technology, the method has excellent qualitative capability, but can still absolutely avoid the interference of matrix effect. Therefore, the experimental example selects the substrate to match the standard solution to establish the standard curve, so as to eliminate or weaken the substrate effect. Taking pork samples as an example, a standard working solution of 128 compounds is prepared by using a pork blank extracting solution, and the standard working solution is subjected to computer analysis and determination after conditions are optimized. And (5) drawing a standard curve by using the peak area (Y) of the compound and the corresponding concentration (X) of the compound, calculating a linear regression equation and a correlation coefficient (r) of the linear regression equation, and calculating LOD and LOQ by using (S/N is 3) and (S/N is 10) respectively. The results show that when the mass concentration is in the interval of 0.5-2000ng/mL, the matrix matching standard curve of 128 veterinary compounds in pork can show good linear relation, and r is more than 0.995. LOD and LOQ are 0.1-20 mug/kg and 0.2-100 mug/kg in sequence, and both meet the experimental requirements. The linear relationship, detection limit and quantification limit of 128 veterinary drugs in pork are shown in table 4.

TABLE 4 Linear relationship, detection limit, quantitation limit of 128 veterinary drugs in pork

(3) Recovery and precision

In the example, blank samples of pork, beef, mutton, chicken, pork liver and chicken liver are respectively added with 128 standard solutions of veterinary drug compounds to prepare positive samples, and the positive samples are repeatedly measured for 6 times under 1-time LOQ, 5-time LOQ and 10-time LOQ according to the established pretreatment method and instrument analysis method, and the recovery rate and the RSD are obtained by solving. The results show that at addition levels ranging from 1-fold LOQ to 10-fold LOQ, process recoveries ranged from 68.7% to 117.1% with Relative Standard Deviation (RSD) ranging from 0.9% to 9.7%. Wherein the pork recovery rate is 68.7-112.5%, and the RSD is 0.9-9.7%; the beef recovery rate is between 70.7 and 108.9 percent, and the RSD is between 1.6 and 9.6 percent; the mutton recovery rate is between 73.7 and 103.6 percent, and the RSD is between 2.1 and 8.8 percent; the chicken recovery rate is between 69.3 and 106.5 percent, and the RSD is between 1.2 and 9.1 percent; the recovery rate of the pork liver is between 71.7 and 117.1 percent, and the RSD is between 1.8 and 9.3 percent; the recovery rate of the chicken liver is 72.1-97.8%, the RSD is 3.6-7.8%, and the example shows that both the accuracy and the precision can meet the experiment requirements. The recovery rate and precision of 128 veterinary drugs in livestock and poultry meat and byproducts are shown in Table 5.

According to the embodiments and experimental examples, the technical scheme provided by the invention realizes the detection of residues of 9 veterinary drug compounds such as male hormone, female hormone, progestogen, glucocorticoid, zearalanol compounds, beta-receptor agonist, sulfonamide antibiotic drugs, quinolone antibiotics and macrolide antibiotics. Compared with other prior art, the veterinary drug compound detected by the invention is more comprehensive in types, more meets the actual application requirements, and has very high application value.

Claims (10)

1. A method for detecting veterinary drug compound residue in livestock meat and byproducts thereof is characterized by comprising the following steps:

(1) preparing a sample to be detected into a test solution;

(2) detecting the test solution by adopting UHPLC-Q/Orbitrap to obtain a chromatogram and a mass spectrum;

(3) obtaining a detection result of the veterinary drug compound residue according to the chromatogram and the mass spectrum;

wherein, the chromatographic conditions in the step (2) are as follows: the chromatographic column adopts C₁₈The column, mobile phase A is aqueous solution containing 0.02-0.1% formic acid and 2.5-20mmol/L ammonium acetate, and mobile phase B is acetonitrile.

2. The detection method according to claim 1, characterized in that: the veterinary drug compound is at least one of androgen, estrogen, progestogen, glucocorticoid, zearalanol compound, beta-receptor stimulant, sulfonamide antibiotic drug, quinolone antibiotic or macrolide antibiotic.

3. The detection method according to claim 2, characterized in that: the veterinary drug compound is selected from dehydrotestosterone, epitestosterone, fluoxymesterone, methyltestosterone, nandrolone propionate, nandrolone, testosterone propionate, trenbolone, meperiandrone, nandrolone phenylpropionate, stanozolol, norandrostenedione, androstenedione, 17 alpha-methylisotosterone, meiandrol, epiandrosterone, 17 beta-hydroxyandrostan-3-one, mesterone, danazol, altrenogest, chlormadinone acetate, levonorgestrel, medroxyprogesterone acetate, megestrol acetate, norelgestromin acetate, norethisterone acetate, 21-alpha-hydroxyprogesterone, norethindrone, 17-alpha-hydroxyprogesterone, beclomethasone propionate, betamethasone, cortisone acetate, hydrocortisone, methylprednisolone, prednisolone, triamcinolone, cortisone, dexamethasone, hydrocortisone acetate, hydrocortisone, medroxyprogesterone acetate, norgestrel, norgestimate, medroxyprogesterone acetate, medroxyprogesterone, medrysone, and, Beclomethasone, flumethasone, budesonide, fluocinonide, fludrocortisone acetate, fluorometholone, clobetasol propionate, aldosterone, prednisone, estriol, estrone, dienestrol, diethylstilbestrol, estradiol, octylphenol, 4-nonylphenol, bisphenol A, ethinylestradiol, alpha-zearalenol, beta-zearalenol, zearalenone, alpha-zearalanol, beta-zearalanol, clenbuterol, ractopamine, chlorpromazine, penbuterol, metaproterenol, formoterol, fenoterol, secobronol, bambuterol, salbutamol, terbutaline, tuobuterol, cimaterol, mabuterol, pyrazolsulfaphenamide, sulfapyridine, sulfacetamide, sulfamethoxydiazine, sulfamethoxazole, sulfamethazine dimethyidipine, sulfamethazine, At least one of sulfamethoxazole, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxine, sulfamodiethoxypyrimidine, sulfamchloropyrazine, sulfamamidine, sulfadiazine, sulfathiazole, sulfamethazine, sulfa nitrobenzene, orbifloxacin, danofloxacin, enrofloxacin, flumequine, fleroxacin, ciprofloxacin, lomefloxacin, nalidixic acid, norfloxacin, fleroxacin, sarafloxacin, difloxacin, sparfloxacin, enoxacin, ofloxacin, erythromycin, kitasamycin, lincomycin, tylosin, tilmicosin, tavermectin, oleandomycin, doramectin, ivermectin, and roxithromycin.

4. The detection method according to claim 1, characterized in that: in the step (1), the method for preparing the test solution comprises the following steps: homogenizing the sample to be detected, extracting with acetonitrile aqueous solution containing formic acid, taking supernatant, performing reverse phase solid phase extraction, blow-drying, and fixing the volume to obtain the product.

5. The detection method according to claim 4, characterized in that: in the step (1), the aqueous acetonitrile solution containing formic acid is an aqueous acetonitrile solution containing 0.2% of formic acid and 80% of volume fraction;

and/or the dosage ratio of the sample to be detected to the acetonitrile aqueous solution containing formic acid is 2g:10 mL;

and/or adding uniform protons, performing vortex oscillation at 2000r/min for 30min, centrifuging at 9500r/min for 5min, and collecting the supernatant;

and/or the solid phase extraction column packing of the reverse phase solid phase extraction is Oasis PRIME HLB;

and/or the amount of the solid phase extraction column packing of the reverse phase solid phase extraction is 3cc 200 mg;

and/or the speed of passing through a solid phase extraction column in the reverse phase solid phase extraction is 1 drop/s;

and/or after constant volume, performing filtration membrane operation on the sample solution, wherein the filtration membrane is a polytetrafluoroethylene membrane.

6. The detection method according to claim 1, characterized in that: in the step (2), the chromatographic conditions further comprise: the chromatographic column is Waters Acquity BEH C₁₈(ii) a And/or, the temperature of the chromatographic column is 30-40 ℃; and/or, flow rate: 0.3-0.5 mL/min; and/or, the sample injection amount is 5-15 mu L;

and/or, a gradient elution procedure:

and/or, the mass spectrometry conditions are:

heating an electrospray ion source; scanning and collecting mode: collecting positive and negative ions simultaneously, and performing primary full scanning/data-dependent secondary scanning; and/or, the spray voltage: 3.5kV (+), -3.0kV (-); and/or, ion transfer tube and heater temperatures: 325 ℃ and 450 ℃; and/or, sheath gas and auxiliary gas flow rates: 35arb, 10 arb; and/or, acquisition range: 100-1000 m/z; and/or, resolution: full MS 75000, dd-MS²17500 (mm); and/or, stemped (n) CE (20%, 40%, 60%); and/or, maximum capacity of ion number in C-Trap: full MS 3X 10⁶、dd-MS² 2×10⁵(ii) a And/or, the maximum injection time in the C-Trap: full MS 100MS, dd-MS² 50ms；

And/or, the tuning correction of the mass axis of the Q/Orbitrap mass spectrum is as follows: injecting the Calmix correction fluid into a mass spectrometer, debugging and observing the characteristic ion peak of the Calmix correction fluid, and automatically correcting by tuning application software when the ion peaks of positive ions and negative ions are stable.

7. The detection method according to claim 1, characterized in that: further comprising the step of establishing a multi-dimensional electronic identity database of standard veterinary drug compounds, said multi-dimensional electronic identity database comprising: basic information, chromatographic information and mass spectrum information of the veterinary drug compound standard;

and (3) comparing the chromatogram and the mass spectrogram with the information in the multi-dimensional electronic identity database to obtain a detection result of the veterinary drug compound residue.

8. The detection method according to claim 7, characterized in that: the basic information comprises Chinese and English names of the compounds, CAS numbers, chemical molecular formulas and theoretical accurate molecular mass numbers;

and/or, the chromatographic information comprises retention time;

and/or the mass spectrum information comprises a parent ion mass number, a fragment ion mass number, an ion addition mode and a standard curve of mass spectrum response area to standard substance concentration.

9. The detection method according to claim 7 or 8, characterized in that: the multidimensional electronic identity database comprises a first-level accurate mass number fingerprint identification database and a second-level HCD fragment ion reference spectrogram confirmation database;

the first-level accurate mass number fingerprint identification database is formed by sequentially recording basic information, chromatographic information and mass spectrum information of the veterinary drug compound in Trace Finder data acquisition and processing software;

the secondary HCD fragment ion reference spectrogram confirmation library is formed by adding and recording secondary HCD fragment ion mass spectrograms obtained by cracking veterinary drug compounds under the optimal collision energy in a high-energy collision pool in mzVault spectrogram management software, wherein the optimal collision energy is the recorded collision energy value CE (20%, 40%, 60% ev) when 3-5 fragment ions with the largest selective ion abundance ratio appear.

10. The detection method according to claim 8, characterized in that: in step (3), the criterion for determining that a certain veterinary drug compound is positive is that the raw data extracted within the 5ppm mass window satisfies all of the following conditions:

1) the retention time error range of the mass spectrum peak of the parent ion is within +/-15 s;

2) the mass number deviation of the parent ions is less than or equal to 5 ppm;

3) the matching number of fragment ions in the secondary mass spectrogram is more than or equal to 2, and the mass number deviation of the fragment ions is less than or equal to 10 ppm.

Images