Method for simultaneously screening 56 drug residues in animal-derived hotpot food materials based on high-resolution mass spectrometry technology

Abstract

The invention belongs to the technical field of mass spectrometric detection, and particularly relates to a method for simultaneously screening 56 drug residues in animal-derived hotpot food materials based on a high-resolution mass spectrometric technique, which comprises the following steps: acquiring a sample, pretreating the sample, preparing a mixed standard working solution, setting database screening, and screening and measuring an actual sample. If the relative deviation between the retention time of a to-be-detected compound in a sample solution and the retention time in a spectrum library is smaller than or equal to + / -2.5% or smaller than or equal to 0.2 min, the signal-to-noise ratio S / N of monitored qualitative ions is larger than or equal to 3, the relative deviation between the precise mass number of parent ions and the theoretical mass number is smaller than or equal to 5 ppm and 1 * 10 <-6 >, and the relative deviation between the precise mass number of a secondary fragment ion is smaller than or equal to 10 ppm, the relative deviation between the precise mass number of the secondary fragment ion is smaller than or equal to 10 ppm and the compound contained in the experiment can be preliminarily judged. According to the method, the sample pretreatment process is simplified, the sample pretreatment operation cost and time are reduced, and meanwhile, the sensitivity and accuracy of hotpot food material matrix analysis are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of mass spectrometry detection, and in particular relates to a method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry technology. Background Art

[0002] Yellow throat, tripe, duck intestines and other special ingredients are deeply loved by hot pot lovers because of their "crisp and tender" and "tender and refreshing" eating characteristics. These hot pot ingredients come from livestock and poultry viscera, organs or aquatic products. Whether in the breeding process, or in the pre-processing process such as swelling, salting, tenderization with processing aids, thawing, pre-packaging, etc., the ingredients may contain non-edible substances such as veterinary drugs, pesticides, human drugs, and drug residues caused by feed and environmental pollution, which brings food safety risks to consumers while enjoying the delicious hot pot.

[0003] In traditional detection methods, the quality and safety issues caused by drug residues in hot pot ingredients are mostly limited to the detection of veterinary drug residues and pesticide residues in a single matrix. In terms of industry standards, the only method standard related to hot pot ingredients is the food supplement inspection method BJS201909 "Determination of quinolone compounds in soy products, hot pot, spicy hot pot and other foods", but the sample matrix is ​​not classified and processed, and the analysis items are extremely limited, and only 11 types of quinolone veterinary drug residues are determined. If the random inspection is based on the category of such food, such as tripe, which is a livestock by-product, nine animal residue items such as furacilin metabolites, chloramphenicol, and sodium pentachlorophenate are sampled. Because yellow throat and duck intestines are not by-products, they are not within the scope of routine random inspections. Therefore, there is a certain risk monitoring blind spot for this type of food.

[0004] Compared with conventional targeted detection, non-targeted screening technology based on high-resolution mass spectrometry has multiple advantages such as high mass accuracy, high resolution, fast scanning speed and high sensitivity. It can match the database according to the mass spectral information such as the exact mass number, secondary fragment ions, retention time, isotope abundance, etc. of the compound, and realize non-targeted rapid screening in the absence of standard controls. It has gradually been used for the discovery and identification of unknown compounds. This technology has been successfully used in rapid non-targeted screening in food safety fields such as organophosphorus pesticide residues in agricultural products and macrolide veterinary drug residues. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for simultaneously screening 56 drug (compound) residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry technology, aiming to discover potential risks that may exist in the ingredients. The detection method is accurate and reliable, simplifies the sample pretreatment process, reduces the sample pretreatment operation cost and time, and improves the sensitivity and accuracy of hot pot ingredient matrix analysis, increases drug residue throughput, and has good application prospects in the qualitative screening of drug residues in animal-derived hot pot ingredients.

[0006] The present invention solves the above technical problems by simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry, comprising the following steps:

[0007] The steps include:

[0008] (1) Pretreatment of samples to be tested:

[0009] Sample extraction: Add extraction solvent to the homogenized sample, then mix, ultrasonically extract and centrifuge;

[0010] Sample purification: Take the extract and add it to the centrifuge tube containing the purifier and dehydrating agent, mix well and centrifuge, take the supernatant and blow dry it with nitrogen at 40℃, add 20% methanol and vortex, pass through 0.22μm filter membrane and measure on the machine;

[0011] (2) Preparation of mixed standard working solution: Pipette the corresponding volume of 56 drug standard stock solutions (100 μg / mL) into a 10 mL volumetric flask, dilute to volume with acetonitrile, and prepare a mixed standard solution for sample testing and membrane filter determination;

[0012] (3) Building a database screening model:

[0013] In full MS-ddMS2 Scan mode, HPLC-electrostatic field orbital hydrazine high-resolution mass spectrometry was used to perform a primary mass spectrometry full scan of the target compounds using 10 μg / L standard substance solutions of 56 drugs. The molecular weight, retention time and fragment ion information of the 56 drugs were obtained by high-resolution mass spectrometry, thereby establishing a chromatographic library and a primary standard library of mass spectra of the 56 drugs.

[0014] (4) Compound screening conditions for actual samples:

[0015] Referring to the high-resolution mass spectrometry confirmation factors for chemical residues proposed by the U.S. FDA Food and Veterinary Drug Steering Committee (OFVM), in full scan and secondary scan modes, the following conditions must be met to be judged as suspected positive results, and extraction is performed within the 5ppm mass window range: ① The target signal response S / N>3. When S / N does not exist, at least 5 consecutive scanning points are required to determine a signal; ② The retention time of the target object is less than 0.2min or within ±2.5% (not more than 0.5min) compared with the retention time parameter in the mass spectrometry library; ③ Compared with the mass spectrometry library, the target object can match the previous parent ion and a secondary fragment ion, and the mass accuracy deviation of the primary parent ion is <5ppm, and the mass accuracy deviation of the secondary fragment ion is <10ppm; If the substance detected in the sample meets the above three conditions at the same time after comparing with the mass spectrometry library, it can be judged as a positive sample.

[0016] In the optimized solution, the extraction solvent is 0.5-1% formic acid acetonitrile.

[0017] In a further optimized solution, the extraction solvent is 0.5% formic acid acetonitrile.

[0018] In a further optimization scheme, a dehydrating agent is added before adding the extraction solvent.

[0019] The dehydrating agent is anhydrous Na2SO 4。

[0020] The dehydrating agent also includes MgSO4, and in terms of mass volume ratio, the MgSO4 dehydrating agent: extracting solution = 50-200:1; in the optimized solution, the MgSO4 dehydrating agent: extracting solution = 50:1.

[0021] The dehydrating agent for initial dehydration is anhydrous Na2SO4. If the sample has a high water content, especially tripe, MgSO4 is used for further dehydration.

[0022] The purifier is C18; in terms of mass-to-volume ratio, the purifier: the extracting solution = 100:2.

[0023] The chromatographic conditions in the step (3) are as follows: chromatographic column: WATERS ACQUITY UPLC HSS T3 (50 mm×2.1 mm1.8 μm); column temperature: 40°C; mobile phase: A is 0.1% formic acid acetonitrile, B is 0.1% formic acid water; flow rate: 0.3 mL / min; injection volume: 10 μL; gradient elution program: 0-4.5 min, 95%-85% B; 4.5-6 min, 85%-5% B; 6-6.5 min, 5% B; 6.5-6.6 min, 5%-95% B; 6.6-8 min, 95% B.

[0024] In the optimization plan, the WATERS ACQUITY HSS T3 chromatographic column was selected, which has a better effect in retaining water-soluble, polar small molecule compounds and has a good peak shape, which is conducive to the separation of non-polar substances such as fats, the main interfering components in the matrix.

[0025] In view of the fact that acetonitrile has a stronger elution ability than methanol, the mobile phase of the present invention selects a water-acetonitrile system, and further investigates the effects of four mobile phases of water-acetonitrile, 0.1% formic acid water-acetonitrile, 0.1% formic acid water-0.1% formic acid acetonitrile, and 0.1% formic acid water (containing 5mmol / L ammonium acetate)-acetonitrile on the chromatographic behavior and sensitivity of 56 compounds. It is found that most target compounds are more stable under acidic conditions and are more conducive to ionization. The acid in the mobile phase can provide the H+ required by the ESI source, thereby improving the ionization efficiency, significantly improving the peak shape of the compound, and improving the sensitivity; after adding ammonium acetate to the mobile phase, not only the peak shape of quinolone and benzimidazole compounds is not significantly improved, but also the mass spectrometry response of quinolone compounds is significantly reduced. Therefore, in order to take into account the peak shape and mass spectrometry response of 56 compounds, 0.1% formic acid water-0.1% formic acid acetonitrile is selected as the mobile phase of this experiment.

[0026] The 56 drug residues are trimethoprim, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine, sulfamethoxazole, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfamethoxazole, sulfadimethoxine, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole ... floxacin, orbifloxacin, oxolinic acid, pefloxacin, cinoxacin, sarafloxacin, sparfloxacin, ciprofloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, flumequine, mebendazole, aminomebendazole, 2-aminoflubendazole, oxfendazole, oxibendazole, 5-hydroxythiabendazole, thiabendazole, albendazole, albendazole sulfone, albendazole sulfoxide, albendazole aminosulfone, fenbendazole, 5-hydroxymebendazole, cambendazole, flubendazole, and fenbendazole sulfone.

[0027] The invention discloses a high-resolution mass spectrometry screening method for drug residues in animal-derived hot pot ingredients. The method utilizes high-performance liquid chromatography-quadrupole / electrostatic field orbital hydrazine high-resolution mass spectrometry and combines with Trace Finder screening software to simultaneously sample 56 drug residues in hot pot ingredients with one needle, thereby realizing high-throughput simultaneous screening. On this basis, the influence of pretreatment conditions on screening compounds is optimized, thereby realizing rapid and high-throughput screening and analysis of drug residues in processed animal-derived hot pot ingredients.

[0028] The optimized QuECHERS purification technology of the present invention greatly simplifies the sample pretreatment steps, making the operation simpler and more economical.

[0029] The present invention solves the problem of qualitative detection of multiple drug residues in hot pot food processed products in the field of food detection. The invention is simple to operate, saves time and effort, has high throughput, and is suitable for daily monitoring of drug residues in animal-derived hot pot food processed products by detection agencies. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention investigates the effects of different extraction solvents on the drug recovery rate in duck intestine.

[0031] Figure 2 The present invention investigates the effects of different extraction solvents on the recovery rate of drugs in Rhizoma Cyperi.

[0032] Figure 3 The present invention investigates the effects of different extraction solvents on the recovery rate of drugs in tripe.

[0033] Figure 4 The present invention investigates the effects of different MgSO4 dosages on drug recovery in duck intestines.

[0034] Figure 5 The present invention investigates the effects of different MgSO4 dosages on the drug recovery rate in Rhizoma Cyperi.

[0035] Figure 6 The present invention investigates the effects of different MgSO4 dosages on the drug recovery rate in tripe.

[0036] Figure 7 The present invention investigates the effects of different C18 dosages on drug recovery in duck intestine.

[0037] Figure 8 The present invention investigates the effects of different C18 dosages on the drug recovery rate in Rhizoma Cyperi.

[0038] Fig. 9 The present invention investigates the effects of different C18 dosages on drug recovery rates in tripe.

[0039] Fig.10 Sulfadiazine R SB / S1 , R S1 / S2 and V cut-of f-value distribution plot.

[0040] Figure 11-Figure 66 The primary extracted ion chromatogram and secondary mass spectrum of 56 drugs (50 ng / mL) in full scan plus automatic triggering secondary scan mode. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments:

[0042] Among them, the instruments:

[0043] Q-Exactive quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system (Thermo Scientific, USA); Vanquish UHPLC ultra-high performance liquid chromatography system (Thermo Scientific, USA); Waters ACQUITY UPLCHSS T3, (50 mm × 2.1 mm, 1.8 μm) liquid chromatography column; VORTEX GENIUS3 vortex oscillator (IKA, Germany); Milli-Q ultrapure water machine (Millipore, USA); MSE225P-ICE-DU electronic balance (Sartorius, Germany); ultrasonic cleaner (Elma, Germany).

[0044] Standards and reagents:

[0045] 57 kinds of standard products were purchased, including 18 quinolone mixed standard solutions in methanol, 23 sulfonamide mixed standard solutions in methanol, and 16 benzamiazole mixed standard solutions in methanol (100 μg / mL, Tianjin Alta Technology Co., Ltd.); one of them had poor mass spectrometry response and recovery under this condition, so it was not included. The standard product information of the following 56 compounds is shown in Table 2. Acetonitrile, methanol (Merck, Germany); formic acid (CNW, Germany) were all chromatographically pure; C18 (40-50 μm) (Agela Technologies); anhydrous sodium sulfate and anhydrous magnesium sulfate (analytical grade, Chongqing Chuandong Chemical Company); Milli-Q ultrapure water was used in the experiment.

[0046] Example 1

[0047] A qualitative screening method for drug residues in animal-derived hot pot ingredients was developed. 56 representative drugs from three categories were selected. By optimizing the extraction conditions and purification technology, ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbital ion trap high-resolution mass spectrometry was combined to perform high-throughput screening of drug residues in animal-derived hot pot ingredients. The specific steps are as follows:

[0048] (1) Sample collection: Tripe, duck intestines, and duck throat samples were collected from hot pot restaurants and other places.

[0049] (2) Sample pretreatment:

[0050] Sample extraction: Weigh 5 g of homogenized sample (accurate to 0.01 g) into a 50 mL stoppered centrifuge tube, add 4 g of anhydrous Na2SO4, vortex mix for 30 seconds, add 10 mL of 0.5% formic acid acetonitrile, vortex mix for 3 minutes, ultrasonically extract for 15 minutes, and centrifuge at 8000 r / min for 3 minutes.

[0051] Sample purification: Take 5 mL of the extract and add it to a centrifuge tube containing 250 mg C18 + 250 mg anhydrous MgSO4, vortex and mix well, then centrifuge at 8000 r / min for 5 min. Take 4 mL of the supernatant and blow dry it with nitrogen at 40°C, add 1 mL of 20% methanol and vortex for 30 seconds, then filter through a 0.22 μm membrane and measure on the machine.

[0052] (3) Solution preparation:

[0053] Preparation of mixed standard working solution: Pipette the corresponding volume of 56 compound standard stock solutions (100 μg / mL) into a 10 mL volumetric flask, dilute to volume with acetonitrile, and prepare mixed standard solutions for sample detection.

[0054] (4) Measure the membrane on the filter machine.

[0055] Chromatographic conditions: chromatographic column: WATERS ACQUITY UPLC HSS T3 (50 mm×2.1 mm 1.8 μm); column temperature: 40°C; mobile phase: A is 0.1% formic acid acetonitrile, B is 0.1% formic acid water; flow rate: 0.3 mL / min; injection volume: 10 μL; gradient elution program: 0-4.5 min, 95%-85% B; 4.5-6 min, 85%-5% B; 6-6.5 min, 5% B; 6.5-6.6 min, 5%-95% B; 6.6-8 min, 95% B.

[0056] The details are shown in Table 1:

[0057] Table 1 Mobile phase gradient elution program

[0058] Time (min) A(％) B(％) 0 5 95 4.5 15 85 6 95 5 6.5 95 5 6.6 5 95 8 5 95 .

[0059] Mass spectrometry conditions: heatable electrospray ion source (HESI), positive and negative ion switching mode; detection method: full scan plus automatic triggering secondary scan (full MS-ddMS 2 ); capillary heating temperature: 320℃; sheath gas (N2) flow rate: 40L / min; auxiliary gas (N2) flow rate: 10L / min; purge gas (N2) flow rate: 3L / min; spray voltage: 3kV; Full MS scan resolution (R): 70000; automatic gain control (AGC target): 3×10 6; Maximum dwell time: 100ms; Scan range: m / z 100-1500; Threshold trigger value: 1×10 5 ; Vertex trigger time: 2~8s; Dynamic exclusion time: 10s; ddMS 2 Scanning resolution (R): 17500; Automatic gain control: 1×10 5 ; Maximum dwell time: 50ms; TopN: 5; Separation window: m / z4.0; Collision energy: (collision energy, NCE) 20, 40 and 60eV respectively; Collision energy step value: 50%.

[0060] (5) Construction of database screening model: construction of chromatographic library and mass spectrum library of 56 drugs;

[0061] In full MS-ddMS 2 In Scan mode, HPLC-ESD high-resolution mass spectrometry was used to perform a full mass spectrometry scan of the target compounds on the prepared standard substance solutions (10 μg / L) of 56 drugs. The molecular weight, retention time and fragment ion information of the 56 drugs were obtained by high-resolution mass spectrometry, thereby establishing a chromatographic library and a primary standard library of mass spectra of 56 veterinary drugs.

[0062] Specifically, a mixed standard solution was prepared, and the sample was injected according to the established chromatographic conditions and mass spectrometry conditions. The Chinese and English names, CAS numbers, and chemical formulas of 56 drugs were accurately input. Trace FinderTM5.0 (Thermo Fisher Scientific, USA) calculated the theoretical mass of each compound, and then data-dependent secondary mass spectrometry scanning (ddMS2) was used while using the dynamic exclusion mode. When the response intensity of the parent ion in the list reached the set threshold, the secondary data was automatically collected; the target was collided at three collision energies (20 / 40 / 60 eV), and the secondary spectra were obtained by summing. The software was used to calculate the precise molecular weight of the main secondary fragments, which was associated with the retention time, precise mass measurement value, Chinese and English name, CAS number, molecular formula and other information of the corresponding compound, and finally the spectral library construction of 56 drugs was completed, as shown in Tables 2 and 3 below. Figure 11-Figure 66 shown.

[0063] Table 2 Database information of 56 drugs

[0064]

[0065]

[0066]

[0067] (6) Qualitative method settings:

[0068] According to EU SANTE 11813 / 2017 regulation, qualitative analysis is based on the retention time of the compound, the accurate mass of the parent ion and the accurate mass of one fragment ion (or the accurate mass of two parent ions). Under the same conditions, if the relative deviation between the retention time of the compound to be tested in the sample solution and the retention time in the library is ≤±2.5% (and no more than 0.5min) or ≤0.2min, the signal-to-noise ratio S / N of the monitored qualitative ion is ≥3, the relative deviation between the accurate mass of the parent ion and the theoretical mass is ≤5ppm (1×10-6), and the relative deviation of the accurate mass of a secondary fragment ion is ≤10ppm, then it can be preliminarily determined that the compound is present in the experiment.

[0069] The target peak area threshold was set to 50 000 to exclude the influence of some impurity peaks and reduce the screening time. The signal-to-noise ratio threshold was set to 10, and the accurate mass deviation was set to 5×10-6. The retention time judgment mode was set to confirm, and the retention time window width was 30s. The fragment ion judgment mode was set to confirm, with a minimum match of 1 fragment ion, a fragment ion intensity threshold of 5 000, and a fragment ion mass deviation of 1×10-5. Isotope screening can be used as supplementary screening information for the target peak. When enabled, the judgment mode is confirm, the match threshold is 90%, the mass deviation is 1×10-5, and the intensity deviation is 20%.

[0070] Example 2

[0071] Method validation experiment:

[0072] The other contents are the same as those in Example 1. For the same sample, three portions are weighed, one portion is not spiked and is recorded as a blank sample; one portion is spiked with 1 times the target drug screening concentration and is recorded as 1×STC; and one portion is spiked with 2 times the target drug screening concentration and is recorded as 2×STC. The STC value of each drug is as follows: Figure 2 The three samples were pre-treated and the blank response ratio (R SB / S1 ) and spike response ratio (R S1 / S2 ), respectively R SB / S1 and R S1 / S2 The value of V cut-off When the sample R S1 / S2 ≤V cut-off , the antibiotic was judged as false negative (FN) in the 1×STC sample; when R SB / S1 ≥V cut-off , the drug was judged as a false positive (FP) in the blank sample.

[0073] R SB / S1= Response value of drug in blank sample / 1×Response value of drug in STC sample (1),

[0074] R S1 / S2 =1×response value of the drug in the STC sample / 2×response value of the drug in the STC sample (2).

[0075] 20 samples of tripe, duck intestines, and yellow throat were selected for method validation. The samples were screened for target drugs using UPLC-HRMS to confirm that the background did not contain the drug to be detected. According to the EU "Guidelines for Validation of Veterinary Drug Residue Screening Methods" method performance evaluation parameters, FP and FN simultaneously meet <5%, which proves that the developed method is suitable for screening this drug in three matrices. According to the response value obtained, the cutoff value V is set cut-off The value is 20% to ensure that there is no overlap between the responses in the blank and spiked samples. Fig.10 When the cutoff value was set to 20%, there was no overlap in the responses of the blank and spiked samples of the 20 samples, thus allowing a clear distinction to be made between the negative (blank) and positive (spiked) samples.

[0076] The cutoff value must ensure a false positive rate of less than 5% and a false negative rate of less than 5%. The false positive is obtained by measuring the response value in the blank sample. When the response value of the blank sample is found to be greater than 20%, it is counted as 1 false positive. The false negative is obtained by measuring the response value in the spiked sample. When the response value of the spiked sample is found to be less than 20%, it is counted as 1 false negative. The results of the method validation are shown in Table 3. It can be seen from the results that the false positive rate and false negative rate of all compounds are less than 5%, which meets the requirements of CRLs 2010 / 01 / 20 for screening results, and the method validation is passed.

[0077] Table 3 Verification results of 56 drugs in tripe

[0078]

[0079]

[0080]

[0081] Experiment 1 Optimization of chromatographic conditions:

[0082] The other contents are as in Example 1. In view of the fact that acetonitrile has a stronger elution ability than methanol, the mobile phase of the present invention selects an acetonitrile system. Under the same conditions, the effects of four mobile phases, namely water-acetonitrile, 0.1% formic acid water-acetonitrile, 0.1% formic acid water-0.1% formic acid acetonitrile, and 0.1% formic acid water (containing 5 mmol / L ammonium acetate)-acetonitrile, on the chromatographic behavior and mass spectrometric response of 56 compounds were further investigated.

[0083] The experiment found that when the mobile phase was water-acetonitrile, the peaks of some quinolone compounds were tailing; after adding formic acid, the peaks of the target compounds were significantly improved, and the sensitivity was also improved. This may be because the acid in the mobile phase provided the H required by the ESI source. + , thereby improving the ionization efficiency; after adding ammonium acetate to the mobile phase, not only did the peak shape of quinolone and benzimidazole compounds not improve significantly, but also the mass spectrometry response of quinolone compounds was significantly reduced.

[0084] Therefore, in order to take into account the peak shape and mass spectrometry response of the 56 compounds, 0.1% formic acid water-0.1% formic acid acetonitrile was selected as the mobile phase of this experiment.

[0085] Experiment 2 Optimization of extraction solvent

[0086] Other contents are as in Example 1. The present invention compares the extraction efficiency of each compound by acetonitrile and methanol. The results show that when acetonitrile is used as an extractant, the overall peak shape of the compound is better, the extraction efficiency is higher, and acetonitrile has better solubility, stronger permeability, and a certain effect of precipitating protein, so acetonitrile is determined as the main extraction solvent. At the same time, considering that the addition of acid in the extraction solvent can both adjust the pH of the system to change the solubility of the compound and improve the extraction efficiency of the compound by destroying the matrix cell tissue structure, under the same conditions, this experiment further investigates the recovery rate of each compound when acetonitrile and 0.1%, 0.2%, 0.5%, 1%, 2%, and 5% formic acid acetonitrile are used as extraction solvents.

[0087] The results are as follows Figure 1-Figure 3 As shown in the figure, the trends of the three matrices of tripe, duck intestine and yellow throat are basically the same. When acetonitrile is used as the extraction agent, the recovery rate of half of the compounds is less than 60%, mainly quinolone compounds; with the increase of formic acid content, the recovery rate of quinolone compounds continues to increase; when the formic acid content reaches 0.5%, the recovery rates of all compounds are between 60% and 120%; when the formic acid content is ≥1%, the recovery rates of sulfonamides and benzimidazoles show a slow downward trend. Therefore, this experiment determined the extraction solvent as 0.5% formic acid acetonitrile.

[0088] Optimization of water removal agent in test 3

[0089] Hot pot ingredients have a very high water content, especially tripe, which has a water content of about 90%. The large amount of water has a great impact on the recovery rate.

[0090] Other contents are as in Example 1. The test found that 2-5g of anhydrous Na2SO4 can remove about 80% of the water in hot pot ingredients. Therefore, the test considers adding a certain amount of anhydrous Na2SO4 to the sample for preliminary water removal, and then further examines the amount of anhydrous MgSO4 added to the extract obtained after sample extraction. Under the same conditions, the test compares the recovery rate of each compound when 0, 50, 100, 200, and 500 mg of anhydrous MgSO4 are added to 1 mL of the extract.

[0091] The results are as follows Figure 4-Figure 6 The results showed that the amount of anhydrous MgSO4 had no significant difference in the recovery rate of each compound in yellow throat and duck intestine, but had a greater impact on tripe. After adding anhydrous MgSO4 to tripe, the recovery rates of sulfonamides and benzimidazole compounds were significantly improved; when the amount of anhydrous MgSO4 was greater than 500 mg, the recovery rate of some quinolone veterinary drugs in tripe dropped to below 50%, which may be due to the high water content of tripe. The strong exothermic phenomenon caused the thermally unstable veterinary drugs to decompose and the recovery rate decreased. When the amount of anhydrous MgSO4 was 50 mg, 100 mg, and 200 mg, the recovery rates of 56 veterinary drugs could reach 70% to 110%. In order to minimize the exothermic reaction, this experiment selected 50 mg of anhydrous MgSO4 as a dehydrating agent for 1 mL of extract.

[0092] Optimization of the purifier in test 4

[0093] PSA and C 18 It is a commonly used purifier in QuECHERS. The other contents are as in Example 1. This experiment investigated PSA and C 18 The effect of purifiers on the recovery of 56 veterinary drugs showed that PSA had a strong adsorption effect on some quinolones and sulfonamides, while C 18 Suitable for removing non-polar and fatty components, so choose C 18 As a purifier. Under the same conditions, the experiment further investigated 50, 100, 200, 300, 500 mg C 18 Purification effect on 2mL extract.

[0094] The results are as follows Figure 7-Figure 9 Shows that when C 18 When the dosage increased to 100 mg, the recovery rates of all compounds increased. 18 When the dosage was greater than 100 mg, the recovery rates of quinolone compounds in the three matrices were significantly reduced, which may be due to a certain adsorption effect. Therefore, the purifier for 2 mL of extract was determined to be 100 mg C. 18 .

[0095] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry, characterized in that: The steps include: (1) Pretreatment of samples to be tested: Sample extraction: Add extraction solvent to the homogenized sample, then mix, ultrasonically extract and centrifuge; Sample purification: Take the extract and add it to the centrifuge tube containing the purifier and dehydrating agent, mix well and centrifuge, take the supernatant and blow dry it with nitrogen, add methanol and vortex, filter the membrane and measure it on the machine; (2) Preparation of mixed standard working solution: Pipette the corresponding volume of 56 drug standard stock solutions into a 10 mL volumetric flask, dilute with acetonitrile, and prepare a mixed standard solution for sample testing and membrane filter determination; (3) Construction of database screening model: In full MS-ddMS2 Scan mode, 10 μg / L standard substance solutions of 56 drugs were prepared and fully scanned by high performance liquid chromatography-electrostatic field orbital hydrazine high-resolution mass spectrometry to obtain the molecular weight, retention time and fragment ion information of the 56 drugs. Thus, the chromatographic library and mass spectrum primary standard library of the 56 drugs were established; (4) Compound screening conditions for actual samples: If the substance detected in the sample meets the following three conditions at the same time after comparison with the mass spectrometry library, it can be judged as a positive sample: Extraction within the 5ppm mass window: ① The target signal response S / N>3, when S / N does not exist, at least 5 consecutive scanning points are required to determine a signal; ② The deviation between the retention time of the target and the retention time parameter in the mass spectrometry library is ≤0.2min or within ±2.5%, not exceeding 0.5min; ③ Compared with the mass spectrometry library, the target matches the previous parent ion and one secondary fragment ion, and the mass accuracy deviation of the primary parent ion is <5ppm, and the mass accuracy deviation of the secondary fragment ion is <10ppm.

2. According to claim 1, a method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry technology is characterized by: The extraction solvent is 0.5-1% formic acid acetonitrile.

3. The method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry according to claim 2, characterized in that: The extraction solvent is 0.5% formic acid acetonitrile.

4. A method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry according to any one of claims 1 to 3, characterized in that: Add the water remover before adding the extraction solvent.

5. The method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry according to claim 4, characterized in that: The dehydrating agent is anhydrous Na2SO4.

6. The method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry according to claim 5, characterized in that: The dehydrating agent also includes MgSO4, and the mass volume ratio is 50-200:

1.

7. The method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry according to claim 1, characterized in that: The purifier is C18; in terms of mass-to-volume ratio, the purifier: the extracting solution = 100:

2.

8. The method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry according to claim 1, characterized in that: The chromatographic conditions in step (3) are as follows: chromatographic column: WATERS ACQUITY UPLCHSS T3, 50 mm×2.1 mm 1.8 μm; Column temperature: 40°C; mobile phase: A is 0.1% formic acid acetonitrile, B is 0.1% formic acid water; flow rate: 0.3 mL / min; injection volume: 10 μL; gradient elution program: 0-4.5 min, 95%-85% B; 4.5-6 min, 85%-5% B; 6-6.5 min, 5% B; 6.5-6.6 min, 5%-95% B; 6.6-8 min, 95% B.

9. A method for simultaneously screening 56 drug residues in animal-derived hot pot ingredients based on high-resolution mass spectrometry, characterized in that: The 56 drug residues are trimethoprim, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine, sulfamethoxazole, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfamethoxazole, sulfadimethoxine, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole ... floxacin, orbifloxacin, oxolinic acid, pefloxacin, cinoxacin, sarafloxacin, sparfloxacin, ciprofloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, flumequine, mebendazole, aminomebendazole, 2-aminoflubendazole, oxfendazole, oxibendazole, 5-hydroxythiabendazole, thiabendazole, albendazole, albendazole sulfone, albendazole sulfoxide, albendazole aminosulfone, fenbendazole, 5-hydroxymebendazole, cambendazole, flubendazole, and fenbendazole sulfone.

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