Method for determining 19 novel veterinary drug residues in animal-derived food based on modified QuEChERS-HPLC-MS / MS method

Abstract

The invention relates to the technical field of food safety detection, and discloses a method for determining 19 novel veterinary drug residues in animal-derived food based on a modified QuEChERS-HPLC-MS / MS (QuEChERS-High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method, and the 19 novel veterinary drug residues in 10 animal-derived matrixes are determined through the QuEChERS-HPLC-MS / MS method. Comprising the following steps: 1) respectively preparing standard stock solutions with different mass concentrations, a mixed standard working stock solution with the mass concentration of 1 mu g / mL and working standard solutions with different mass concentrations by using a standard substance; (2) adding a proper amount of the mixed standard working stock solution into the extracted and purified blank sample residues, preparing a series of matrix matching mixed standard solutions with different concentrations, filtering, determining through HPLC-MS / MS, and drawing a blank matrix standard curve; and (3) homogenizing the taken cattle, pig, sheep, horse, chicken, fish, shrimp, liver, milk and fat samples, extracting and purifying the homogenized samples, and performing HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) on-machine analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a method for determining 19 new veterinary drug residues in animal-derived foods based on a modified QuEChERS-HPLC-MS / MS method. Background Art

[0002] As the global demand for animal-based foods continues to grow, concerns about the safety risks of veterinary drug residues have also increased. In recent years, the misuse and abuse of veterinary drugs to improve the growth and feed efficiency of livestock have become common. Veterinary drug residues refer to toxicologically significant drug prototypes, metabolites, and drug impurities that accumulate or store in the edible parts (including cells, tissues, or organs) of edible animals, or enter eggs or mammalian milk. The presence of drug residues in food is generally attributed to the use of veterinary drugs in animal husbandry. These drugs tend to accumulate in liver and kidney tissues, and long-term intake can have toxic effects on the human body, such as carcinogenesis, organ dysfunction, drug resistance and other adverse consequences. Therefore, many countries have promulgated relevant regulations and standards on maximum residue limits (MRLs) of veterinary drugs (European).

[0003] There are many veterinary drug residues in animal-derived foods, and the relevant standards and specifications are updated rapidly. Countries that have not yet established the necessary regulations and standards always face potential technical challenges in terms of detection technology. In order to overcome technical barriers to international trade, a substance screening work was carried out based on the new monitoring indicators (including new substances, new limits, new matrices, etc.) included in the recent amendments to food safety regulations in countries such as the European Union (EU), South Korea, Canada, and the United States (US). 19 new animal residue compounds for which my country has not yet established relevant technical standards were screened out, including (1) yohimbine hydrochloride, a topical preservative (MRL: 0.01 mg / kg, source: South Korea); (2) sulfaethoxypyridazine, a sulfonamide antibacterial agent (MRL: 0.01 mg / kg, source: Canada); (3) tripelennamine hydrochloride, an antipruritic (MRL: 0.2 mg / kg, source: the United States), etc.

[0004] Most pretreatment and purification methods for veterinary drug residues are based on solid phase extraction (SPE), dispersed solid phase extraction (DSPE), magnetic solid phase extraction (M-SPE), dispersed liquid-liquid microextraction (DLLME) and liquid-liquid extraction (LLE) techniques. At present, solid phase extraction columns are widely used, but a series of operations such as column activation, loading, washing, and elution in pretreatment are time-consuming and require a large amount of reagents. Therefore, QuEChRES-DSPE is proposed as a pretreatment cleanup material. This method is simple to operate, uses less solvent, effectively avoids sieve plate clogging caused by complex matrices, and improves the pretreatment and purification efficiency of large-volume samples. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining 19 new veterinary drug residues in animal-derived foods based on a modified QuEChERS-HPLC-MS / MS method, and to determine 19 new veterinary drug residues in 10 animal-derived matrices by QuEChERS combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0006] The present invention is achieved by the following technical scheme: a method for determining 19 new veterinary drug residues in animal-derived foods based on a modified QuEChERS-HPLC-MS / MS method, comprising the following steps:

[0007] 1) Preparation of standard stock solutions: Standard stock solutions of different mass concentrations, mixed standard working stock solutions with a mass concentration of 1 μg / mL, and working standard solutions of different mass concentrations were prepared using standard products (new veterinary drug residues);

[0008] 2) Preparation of blank matrix standard curve: Take an appropriate amount of mixed standard working stock solution and add it to the blank sample residue after extraction and purification to prepare a series of matrix-matched mixed standard solutions of different concentrations. After filtering, measure by HPLC-MS / MS, and then draw a blank matrix standard curve;

[0009] 3) Sample preparation: The cattle, pigs, sheep, horses, chickens, fish, shrimps, liver, milk, and fat samples were homogenized, and the homogenized samples were extracted and purified, and then analyzed by HPLC-MS / MS.

[0010] To further implement the present invention better, the step 1) includes the following specific steps:

[0011] 1.1) Accurately weigh 0.01 g of each of ciclesonide standard and butaquinate standard into a 10 mL volumetric flask, add acetonitrile and methanol to dissolve and dilute the two standards to the scale, prepare a single standard ciclesonide stock solution and a single standard butaquinate stock solution with a mass concentration of 1000 μg / mL, and store at -20°C in the dark;

[0012] 1.2) Each standard stock solution of the remaining 17 liquid standards was prepared as follows: 1 mL of the standard solution to be prepared was accurately transferred to a 10 mL volumetric flask, and methanol or acetonitrile was added to reach a final volume of 10 mL. Then, a standard stock solution with a mass concentration of 10 μg / mL or 100 μg / mL was prepared and stored at -20°C in a dark environment for use;

[0013] 1.3) Accurately weigh appropriate amounts of 19 standard stock solutions, dilute with methanol, and prepare a mixed standard working stock solution with a mass concentration of 1 μg / mL;

[0014] 1.4) Prepare a series of working standard solutions with concentrations of 0.01 ng / mL, 0.1 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL and 50.0 ng / mL.

[0015] To further implement the present invention better, the step 2) is specifically as follows:

[0016] 2.1) Take an appropriate amount of the mixed standard working stock solution into the residues of 8 blank samples after extraction and purification, and blow dry with nitrogen at 55°C;

[0017] 2.2) Add 1 mL of methanol, vortex to dissolve, and prepare a matrix-matched series of mixed standard solutions with concentrations of 0.01, 0.1, 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 ng / mL, respectively. Pass through a 0.22 μm filter membrane and apply to the machine for HPLC-MS / MS determination;

[0018] 2.3) Draw a blank matrix standard curve with the measured characteristic ion peak area as the ordinate and the corresponding standard solution concentration as the abscissa.

[0019] To further implement the present invention, the method for preparing the blank sample comprises:

[0020] 2.1.1) For blank samples of cattle, sheep, horse, chicken, fish, shrimp, liver, milk and fat:

[0021] 2.1.1.1) Accurately weigh 2.0 g of a single sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 minutes to allow the solution to fully penetrate into the tissue cells (the fat sample is heated in a 60°C water bath until it melts);

[0022] 2.1.1.2) After step 2.1.1.1), add 1 g NaCl to the milk sample, add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the remaining samples, and shake and extract for 5 min; preferably, only add 10 mL 85% acetonitrile to the remaining samples, and shake and extract for 5 min;

[0023] 2.1.1.3) After step 2.1.1.2), extract the sample in an ultrasonic water bath for 15 min, and centrifuge at 10,000 rpm at room temperature (20±2°C) or 4°C or -4°C for 5 min; preferably, centrifuge at 10,000 rpm at 4°C for 5 min;

[0024] 2.1.1.4) The supernatant obtained in step 2.1.1.3) is added to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortexed for 2 min, and centrifuged at 10000 r / min and 4°C for 5 min; preferably, the supernatant obtained in step 2.1.1.3) is added to a purification tube containing 100 mg of C18 and 1100 mg of MgSO4, vortexed for 2 min, and centrifuged at 10000 r / min and 4°C for 5 min;

[0025] 2.1.1.5) After step 2.1.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow with nitrogen at 45°C until it is completely dry;

[0026] 2.1.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain blank samples of cattle, sheep, horse, chicken, fish, shrimp, liver, milk and fat;

[0027] 2.1.2) For pig blank samples:

[0028] 2.1.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells;

[0029] 2.1.2.2) After step 2.1.2.1), add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the pig muscle sample and shake and extract for 5 minutes; preferably, add 10 mL 85% acetonitrile to the pig muscle sample and shake and extract for 5 minutes;

[0030] 2.1.2.3) After step 2.1.2.2), the sample is extracted in an ultrasonic water bath for 15 minutes, and centrifuged at 10,000 rpm at room temperature (20±2°C) or 4°C or -4°C for 5 minutes; preferably, centrifuged at 10,000 rpm at 4°C for 5 minutes;

[0031] 2.1.2.4) Mix the supernatant obtained in step 2.1.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 rpm at 4°C for 5 min;

[0032] 2.1.2.5) After step 2.1.2.4), the acetonitrile layer is transferred to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortexed for 2 min, and centrifuged at 10000 r / min at 4°C for 5 min; preferably, the acetonitrile layer is transferred to a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortexed for 2 min, and centrifuged at 10000 r / min at 4°C for 5 min;

[0033] 2.1.2.6) After step 2.1.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow nitrogen at 45°C until it is completely dry;

[0034] 2.1.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain a pig muscle blank sample.

[0035] To further implement the present invention, the homogenized sample is extracted and purified, comprising the following steps:

[0036] 3.1) For samples of cattle, sheep, horses, chicken, fish, shrimp, liver, milk and fat:

[0037] 3.1.1) Accurately weigh 2.0 g of sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells (the fat sample is heated in a 60°C water bath until it melts);

[0038] 3.1.2) After step 3.1.1), add 1 g NaCl to the milk sample, add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the remaining samples, and shake and extract for 5 min; preferably, add 10 mL 85% acetonitrile to the remaining samples, and shake and extract for 5 min;

[0039] 3.1.3) After step 3.1.2), the sample is extracted in an ultrasonic water bath for 15 minutes, and centrifuged at 10,000 rpm at room temperature (20±2°C) or 4°C or -4°C for 5 minutes; preferably, centrifuged at 10,000 rpm at 4°C for 5 minutes;

[0040] 3.1.4) The supernatant obtained in step 3.1.3) is added to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortexed for 2 min, and centrifuged at 10000 r / min and 4°C for 5 min; preferably, the supernatant obtained in step 3.1.3) is added to a purification tube containing 100 mg of C18 and 1100 mg of MgSO4, vortexed for 2 min, and centrifuged at 10000 r / min and 4°C for 5 min;

[0041] 3.1.5) After step 3.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow with nitrogen at 45°C until it is completely dry;

[0042] 3.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis;

[0043] 3.2) For pig samples:

[0044] 3.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells;

[0045] 3.2.2) After step 3.2.1), add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the pig muscle sample and extract with shaking for 5 minutes; preferably, add 10 mL 85% acetonitrile to the pig muscle sample and extract with shaking for 5 minutes;

[0046] 3.2.3) After step 3.2.2), the sample is extracted in an ultrasonic water bath for 15 minutes, and centrifuged at 10,000 rpm at room temperature (20±2°C) or 4°C or -4°C for 5 minutes; preferably, centrifuged at 10,000 rpm at 4°C for 5 minutes;

[0047] 3.2.4) The supernatant obtained in step 3.2.3) was mixed with 5 ml of acetonitrile-saturated n-hexane, vortexed for 2 min, and then centrifuged at 10,000 rpm at 4°C for 5 min;

[0048] 3.2.5) After step 3.2.4), the acetonitrile layer is transferred to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortexed for 2 min, and centrifuged at 10000 r / min at 4°C for 5 min; preferably, the acetonitrile layer is transferred to a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortexed for 2 min, and centrifuged at 10000 r / min at 4°C for 5 min;

[0049] 3.2.6) After step 3.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45°C;

[0050] 3.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis.

[0051] To further implement the present invention, when conducting HPLC-MS / MS analysis,

[0052] The chromatographic conditions are:

[0053] The chromatographic columns ACQUITY UPLC HSS T3 or ACQUITY UPLC BEH C18 were used for separation; the mobile phase A was methanol or acetonitrile, and the mobile phase B was 0.1% HCOOH, 0.2% HCOOH, 5mmol / L HCOONH4, 5mmol / L CH3COONH4 or 5mmol / L CH3COONH4+0.1%HCOOH; the elution conditions of the veterinary drug residues are: 5% mobile phase A, 0-2min; 5-50% mobile phase A, 2-2.5min; 50-85% mobile phase A, 2.5-6min; 85-90% mobile phase A, 2.5-6min; 90% mobile phase A at 9-10min; 90-95% mobile phase A at 10-10.1min, 95% mobile phase A at 10.1-12min, 95-5% mobile phase A at 12-12.5min, 5% mobile phase A at 12.5min-15min, and the total duration of the experiment is 15min. In addition, the flow rate is 0.25mL / min and the injection volume is 3.0μL;

[0054] Testing conditions:

[0055] Mass spectrometry was performed using a triple quadrupole mass spectrometer in electrospray positive ionization mode with:

[0056] The electrospray capillary voltage was 3.00 kV, and the ESI+cone voltage was 30 V; the dissolved gas flow rate was 1000 L / h, the collision gas flow rate was set to 0.15 mL / min, and the temperature was 200-500 °C. Qualitative analysis of each compound was performed under the multiple reaction monitoring (MRM) mode.

[0057] To further better implement the present invention, a chromatographic column ACQUITY UPLC HSS T3 was used, the mobile phase A was methanol, and the mobile phase B was 0.2% HCOOH; and the triple quadrupole mass spectrometer was a Waters Quattro Premier triple quadrupole mass spectrometer.

[0058] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0059] The present invention is designed based on the new veterinary drug compound limit standard regulations promulgated in recent years by South Korea, Canada, the United States and the European Union, and my country has not yet established relevant detection standards. The sample is extracted with 85% acetonitrile, separated by ACQUITY UPLC HSS T3 column (2.1mm*100mm, 1.8μm), and gradient eluted with methanol-0.2% formic acid water as the mobile phase. The detection of the analyte is achieved by using positive ion electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode, and quantification is performed by matrix matching external standard method.

[0060] When the present invention is implemented, the target veterinary drug residues in ten matrices have good linearity, ranging from 0.01-50.0 ng / mL (R 2 >0.991). The detection limit (LOD) is 0.01-1.29 μg / kg, and the quantification limit (LOQ) is 0.02-4.31 μg / kg. The recovery rate is between 60.3% and 118.5%, and the relative standard deviation (RSD) is ≤20.9%. The present invention is simple and highly sensitive, and meets the maximum limit requirements specified in the relevant standards of South Korea, Canada, the United States and the European Union. The present invention is suitable for the rapid screening, qualitative and quantitative analysis of substances such as metoimidazole, guaifenesin, and methylephedrine hydrochloride in animal-derived foods.

[0061] The present invention develops a method combining QuEChRES with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) for the determination of 19 new veterinary drug residues in animal-derived foods. The method can simultaneously detect multiple new veterinary drug residues in multiple matrices (beef, pork, sheep, horse, chicken, shrimp, fish, liver, milk, fat). Through the selection and optimization of chromatographic conditions, chromatographic columns, extraction solvents, centrifugal temperatures and purification powders, the 19 target compounds showed a good linear relationship (R2>0.991) in the range of 0.01-50.0 ng / mL. The average recovery rate of ten matrices ranged from 60.3-118.5%, with RSD≤20.9%. The method is simple, rapid and highly sensitive, and provides reliable technical support for the extraction and determination of 19 new veterinary drugs in multi-matrix animal-derived foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 There are 19 new primary residues (50 ng / ml) separated on the chromatogram.

[0063] Figure 2a A diagram showing the effect of optimizing the extraction solvent on the recovery rate of 19 veterinary drug residues (taking fish as an example).

[0064] Figure 2b A diagram showing the effect of optimizing the extraction solvent on the recovery rate of 19 veterinary drug residues (taking shrimp as an example).

[0065] Figure 2c This figure shows the effect of optimizing the extraction solvent on the recovery rate of 19 veterinary drug residues (taking chicken as an example).

[0066] Figure 2d This is a diagram showing the effect of optimizing the extraction solvent on the recovery rate of 19 veterinary drug residues (taking pigs as an example).

[0067] Figure 2e This is a diagram showing the effect of optimizing the extraction solvent on the recovery rate of 19 veterinary drug residues (taking cattle as an example).

[0068] Figure 3 Schematic diagram of the recovery rates of 19 new veterinary drug residues (taking grass carp as an example) at different optimized centrifugation temperatures.

[0069] Figure 4 Schematic diagram of the recovery rate of veterinary drug residues in different types of purified powders (taking liver as an example).

[0070] Figure 5a C18 dose optimization for 19 veterinary drug residues in beef matrix.

[0071] Figure 5b C18 dose optimization for 19 veterinary drug residues in 9 matrices other than beef.

[0072] Figure 6 Schematic diagram of the optimization of anhydrous magnesium sulfate (MgSO4) for 19 veterinary drug residues in 10 matrices.

[0073] Figure 1 In the figure, a. ethodicarbamazine; b. racemic methylephedrine hydrochloride; c. ormeprim; d. acridine-3,6-diamine; e. pentylenetetrazol; f. yohimbine; g. 3,6-diamino-10-methylacridine chloride; h. tripeliramine; i. antipyrine; j. sulfathioxazine; k. anthranilic acid; l. guaifenesin; m. acetanilide; n. rivanol; o. phenacetin; p. medetomidine; q. loperamide; r. dehydrocholic acid; s. butyquinate; t. ciclesonide, the vertical axis is the response rate (Response).

[0074] Figure 2 (ae), Figure 3 The vertical axis is the recovery rate.

[0075] Figure 4 Medium Group1: 80mg C18+1000mg MgSO4+80mg PSA; Group2: 80mg C18+1000mgMgSO4+40mg GCB; Group3: 80mg C18+1000mg MgSO4+80mg PSA+40mg GCB; Group4: 80mg C18+1000mg MgSO4.

[0076] Among them, the 10 matrices are: cattle, pigs, sheep, horses, chickens, fish, shrimps, liver, milk, and fat. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0078] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0079] Glossary:

[0080] In the drawings given in the present invention, Acetanilide is acetanilide, o-Aminobenzoic acid (Anthranilic acid) is o-aminobenzoic acid, Pentetrazol (Pentylenetetrazol) is pentylenetetrazol, Methyl ephedrine hydrochloride (dl-Methylephedrine HCl) is racemic methylephedrine hydrochloride, Phenacetin is phenacetin, Antipyrine (Phenazone) is antipyrine, Guaifenesin is guaifenesin, Diethylcarbamazine is diethylcarbamazine, * Acriflavine (8048-52-0) is a mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-Acridinediamine (Proflavine), Metomidate is metomidate, Ethacridine lactate monohydrate (Acrinol) is levonol, Tripelennamine hydrochloride is tripelennamine, Ormetoprim is ormetoprim, Sulfaethoxypyridazine is sulfaethoxypyridazine, Yohimbine hydrochloride is yohimbine, Buquinolate is buquinolate, Dehydrocholic acid is dehydrocholic acid, Loperamide hydrochloride is loperamide, Ciclesonide is ciclesonide, Acetonitrile is acetonitrile, Ethyl acetate is ethyl acetate, 0.1% formic acid acetonitrile is 0.1% formic acid acetonitrile, and 1% ammonia acetonitrile is 1% ammonia acetonitrile.

[0081] In order to achieve better technical effects, the present invention involves the following instruments, materials and reagents:

[0082] HPLC system (HPLC 1-class-XEVOTQ-XS, Waters Corporation, USA); chromatographic column Acquity UPLC HSS T3 (2.1 mm*100 mm, 1.8 μm, Waters Corporation, USA).

[0083] Methanol (MeOH) and acetonitrile (CAN) were of LC-MS grade and purchased from CNW Technologies GmbH (Düsseldorf, Germany). Acetic acid (CH3COONH4), ammonium formate (HCOONH4), formic acid (HCOOH), and ammonium hydroxide (NH3·H2O) were of HPLC grade and purchased from Chengdu Kelong Chemical Co., Ltd. (Sichuan, China). Analytical grade sodium chloride (NaCl), C18 adsorbent (40-63 μm, C18), anhydrous sodium sulfate (Na2SO4), and anhydrous magnesium sulfate (MgSO4) were purchased from Anpel Laboratory Technologies (Shanghai, China). In addition, all water used in the analysis was from the Mili-Q Integral 3 ultrapure water unit manufactured by Millipore Corporation (Boston, USA).

[0084] Butaquinate (10 mg, 98.3%, CAS 5486-03-3) and ciclesonide (100 mg, 99.3%, CAS 126544-47-6) were purchased from Beijing Manhag Biotechnology Co., Ltd. (Beijing, China). Other standards were all liquid standard materials with a purity of ≥94.6% from Alta science (Tianjin, China). Table 1 provides more detailed information about the 19 new veterinary drug residue standards, including CAS number, molecular weight, solvent, etc. In particular, acridinium is a mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-Acridinediamine (Proflavine). The two substances showed different retention times on the chromatographic column. Therefore, this substance was considered as two separate substances in the following analysis.

[0085] Table 1 CAS number, molecular weight, solvent, chemical structure and pharmacological use of 19 veterinary residues

[0086]

[0087]

[0088] *Commercially available acridinium is a mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-Acridinediamine (Proflavine).

[0089] Embodiment 1:

[0090] The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method includes the following steps:

[0091] 1) Preparation of standard stock solutions: Standard stock solutions of different mass concentrations, mixed standard working stock solutions with a mass concentration of 1 μg / mL, and working standard solutions of different mass concentrations were prepared using standard products (new veterinary drug residues);

[0092] 2) Preparation of blank matrix standard curve: Take an appropriate amount of mixed standard working stock solution and add it to the blank sample residue after extraction and purification to prepare a series of matrix-matched mixed standard solutions of different concentrations. After filtering, measure by HPLC-MS / MS, and then draw a blank matrix standard curve;

[0093] 3) Sample preparation: The cattle, pigs, sheep, horses, chickens, fish, shrimps, liver, milk, and fat samples were homogenized, and the homogenized samples were extracted and purified, and then analyzed by HPLC-MS / MS.

[0094] Among them, cattle (muscle), pig (muscle), sheep (muscle), horse (muscle), chicken (muscle), fish (grass carp, skin, muscle), shrimp (muscle), liver (pork liver), milk, and fat (lard) samples can be taken from local markets or supermarkets. Each sample is homogenized in a homogenizer and stored at -20°C for future use.

[0095] Among them, 19 kinds of standard substances (new veterinary drug residues) are: ethodicarbamazine, acriflavine (8048-52-0), racemic methylephedrine hydrochloride, ormeprim, pentylenetetrazol, yohimbine, tripelennamine, antipyrine, sulfathioxazine, anthranilic acid, guaifenesin, acetanilide, rivanol, phenacetin, medetomidine, loperamide, dehydrocholic acid, butyquinate, ciclesonide; among them, acriflavine (8048-52-0) is a mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-Acridinediamine (Proflavine). The two have different retention times on the chromatographic column, so the present invention will analyze and discuss them separately during the experiment.

[0096] Embodiment 2:

[0097] This embodiment is further optimized on the basis of the above embodiment, and the same points as the above technical solution are not repeated here. The step 1) includes the following specific steps:

[0098] 1.1) Accurately weigh 0.01 g of each of ciclesonide standard and butaquinate standard into a 10 mL volumetric flask, add acetonitrile and methanol to dissolve and dilute the two standards to the scale, prepare a single standard ciclesonide stock solution and a single standard butaquinate stock solution with a mass concentration of 1000 μg / mL, and store at -20°C in the dark;

[0099] 1.2) The standard stock solutions of each of the remaining 17 liquid standards (diethylcarbamazine, *acridin, racemic methylephedrine hydrochloride, ormeprim, pentylenetetrazol, yohimbine, tripelennamine, antipyrine, sulfathioxazine, anthranilic acid, guaifenesin, acetanilide, rivanol, phenacetin, medetomidine, loperamide, dehydrocholic acid) were prepared as follows: 1 mL of the standard solution to be prepared was accurately transferred to a 10 mL volumetric flask, and methanol or acetonitrile was added to reach a final volume of 10 mL, and then a standard stock solution with a mass concentration of 10 μg / mL or 100 μg / mL was prepared, and stored at -20°C in a dark environment for use;

[0100] 1.3) Accurately weigh appropriate amounts of 19 standard stock solutions, dilute with methanol, and prepare a mixed standard working stock solution with a mass concentration of 1 μg / mL;

[0101] 1.4) Prepare a series of working standard solutions with concentrations of 0.01 ng / mL, 0.1 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL and 50.0 ng / mL.

[0102] Embodiment 3:

[0103] This embodiment is further optimized on the basis of any of the above embodiments, and the same points as the above technical solutions are not repeated here. The step 2) is specifically as follows:

[0104] 2.1) Take an appropriate amount of the mixed standard working stock solution into the residues of 8 blank samples after extraction and purification, and blow dry with nitrogen at 55°C;

[0105] 2.2) Add 1 mL of methanol, vortex to dissolve, and prepare a matrix-matched series of mixed standard solutions with concentrations of 0.01, 0.1, 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 ng / mL, respectively. Pass through a 0.22 μm filter membrane and apply to the machine for HPLC-MS / MS determination;

[0106] 2.3) Draw a blank matrix standard curve with the measured characteristic ion peak area as the ordinate and the corresponding standard solution concentration as the abscissa.

[0107] Embodiment 4:

[0108] This embodiment is further optimized on the basis of any of the above embodiments, and the same points as the above technical solutions are not repeated here. The preparation method of the blank sample includes:

[0109] 2.1.1) For blank samples of cattle, sheep, horse, chicken, fish, shrimp, liver, milk and fat:

[0110] 2.1.1.1) Accurately weigh 2.0 g of a single sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 minutes to allow the solution to fully penetrate into the tissue cells (the fat sample is heated in a 60°C water bath until it melts);

[0111] 2.1.1.2) After step 2.1.1.1), add 1 g NaCl to the milk sample and add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the remaining samples and extract by shaking for 5 min;

[0112] 2.1.1.3) After step 2.1.1.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature (20 ± 2°C) or 4°C or -4°C;

[0113] 2.1.1.4) The supernatant obtained in step 2.1.1.3) was added to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortexed for 2 min, and centrifuged at 10000 r / min and 4°C for 5 min;

[0114] 2.1.1.5) After step 2.1.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow with nitrogen at 45°C until it is completely dry;

[0115] 2.1.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain blank samples of cattle, sheep, horse, chicken, fish, shrimp, liver, milk and fat;

[0116] 2.1.2) For pig blank samples:

[0117] 2.1.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells;

[0118] 2.1.2.2) After step 2.1.2.1), add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the pig muscle sample and extract with shaking for 5 min;

[0119] 2.1.2.3) After step 2.1.2.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature (20 ± 2°C) or 4°C or -4°C;

[0120] 2.1.2.4) Mix the supernatant obtained in step 2.1.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 rpm at 4°C for 5 min;

[0121] 2.1.2.5) After step 2.1.2.4), transfer the acetonitrile layer to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortex for 2 min, and centrifuge at 10000 r / min at 4°C for 5 min;

[0122] 2.1.2.6) After step 2.1.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow nitrogen at 45°C until it is completely dry;

[0123] 2.1.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain a pig muscle blank sample.

[0124] Embodiment 5:

[0125] This embodiment is further optimized on the basis of any of the above embodiments, and the same points as the above technical solutions are not repeated here. The preparation method of the blank sample includes:

[0126] 2.1.1) For blank samples of cattle, sheep, horse, chicken, fish, liver, shrimp, milk and fat:

[0127] 2.1.1.1) Accurately weigh 2.0 g of a single sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 minutes to allow the solution to fully penetrate into the tissue cells (the fat sample is heated in a 60°C water bath until it melts);

[0128] 2.1.1.2) After step 2.1.1.1), add 1 g NaCl to the milk sample and 10 mL 85% acetonitrile to the remaining samples, and extract by shaking for 5 min;

[0129] 2.1.1.3) After step 2.1.1.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm and 4°C for 5 min;

[0130] 2.1.1.4) Add the supernatant obtained in step 2.1.1.3) into a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex mix for 2 min, and centrifuge at 10000 rpm and 4°C for 5 min;

[0131] 2.1.1.5) After step 2.1.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow with nitrogen at 45°C until it is completely dry;

[0132] 2.1.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain blank samples of cattle, sheep, horse, chicken, fish, shrimp, liver, milk and fat;

[0133] 2.1.2) For pig blank samples:

[0134] 2.1.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells;

[0135] 2.1.2.2 After step 2.1.2.1), add 10 mL of 85% acetonitrile to the pig muscle sample and extract with shaking for 5 min;

[0136] 2.1.2.3) After step 2.1.2.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm and 4°C for 5 min;

[0137] 2.1.2.4) Mix the supernatant obtained in step 2.1.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 rpm at 4°C for 5 min;

[0138] 2.1.2.5) After step 2.1.2.4), transfer the acetonitrile layer to a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex for 2 min, and centrifuge at 10000 rpm at 4°C for 5 min;

[0139] 2.1.2.6) After step 2.1.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow nitrogen at 45°C until it is completely dry;

[0140] 2.1.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain a pig muscle blank sample.

[0141] In order to achieve good results, the extraction of samples (and blank samples) was optimized accordingly:

[0142] 1. Extraction solvent:

[0143] Extraction reagents have an important influence on the recovery rate, target peak appearance time and peak shape of several veterinary drug residues. Commonly used extraction solvents in animal-derived foods include methanol, acetonitrile and ethyl acetate. At the same time, the pH value of the extraction environment may also affect the extraction efficiency of the target substance. The present invention selects acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile, and 1% ammonia acetonitrile as extraction solvents to compare the extraction effects. Figure 2a The results showed that the extraction efficiency was higher when acetonitrile was used as the extraction solvent in the fish matrix. Although the recovery of phenacetin in 1% ammonia acetonitrile, antipyrine and sulfamethoxypyridazine in 0.1% formic acid acetonitrile as extraction solvents was slightly higher than that of acetonitrile, the above conclusions remain unchanged. Taking shrimp as an example ( Figure 2b ), when 0.1% formic acid acetonitrile was used as the extraction solvent, the recoveries of anthranilic acid, antipyrine, 3,6-acridinediamine, tripelennamine and ciclesonide were good, but butaquinate, ormeprim, methylephedrine and yohimbine were not suitable for extraction in an acidic environment, and the recoveries were less than 30%. The recoveries of each substance extracted with acetonitrile were relatively stable, while the recoveries of 1% ammonia acetonitrile extraction varied greatly. In chicken ( Figure 2c ), extraction of anthranilic acid, ormeprim and butaquinate with 1% ammonia in acetonitrile showed very high recoveries (up to 120% or more). On the other hand, the recoveries of acetanilide, methylephedrine hydrochloride, medetomidine, antipyrine and loperamide were lower (<50%). When extracted with 0.1% formic acid in acetonitrile, the recovery efficiency of phenacetin, ethopropazine, ormeprim and loperamide was higher than that of acetonitrile, while the recovery efficiency of other substances was higher under acetonitrile conditions. Ethyl acetate limited the recovery of all substances to a maximum of 40%. In addition, when extracting anthranilic acid from pork, 1% ammonia in acetonitrile showed an excessively high recovery ( Figure 2d ). The recovery rates of antipyrine, tripelennamine, and ciclesonide when extracted with 0.1% formic acid acetonitrile were higher than those of acetonitrile, and the recovery rates of the remaining substances were higher when extracted with acetonitrile. The recovery rates of phenacetin, ethopropazine, rivanol, and loperamide were comparable in the two solvents. In addition, the recovery advantage of acetonitrile as a beef extraction solvent was more significant ( Figure 2e). The recovery rate of ethyl acetate was also the worst, which was hardly higher than 20%. Since animal matrices contain a large amount of lipids and many lipophilic compounds are easily extracted by ethyl acetate, the matrix interference is high, resulting in low extraction efficiency. The easy emulsification of ethyl acetate as an extraction solvent is also another possible factor. Acetonitrile has good solubility and strong penetration, which can precipitate proteins and effectively avoid excessive extraction of lipids, so it is more advantageous in most matrices. However, proteins are prone to agglutination in organic solvents, and acetonitrile extraction did not obtain satisfactory recovery rates in the above ten matrices. Adding an appropriate amount of pure water to the extractant can reduce the proportion of acetonitrile, promote sample dispersion and slow down uniform agglomeration. In addition, a mixture of acetonitrile and water can extract analytes in a variety of polarity ranges from the matrix. Therefore, the experiment continued to compare the extraction efficiency of 80%, 85%, 90% and 95% acetonitrile in Figure 2 (a~e). The results show that 85% acetonitrile has good recovery rates for the target substances in all studied matrices, especially for butyquinate, which has a significant enhancement effect, especially in pork, beef and shrimp matrices. Subsequent optimization treatment can also improve the sensitivity of the method. Therefore, 85% acetonitrile aqueous solution was optimally selected as the extraction solvent.

[0144] Furthermore, the high fat content of pork requires the implementation of an additional extraction step. Animal fats are primarily composed of long-chain saturated fatty acids, which are characterized by the presence of two groups: a polar carboxyl group and a non-polar hydrocarbon group. The solubility of fatty acids in water is affected by the length of the hydrocarbon group, with longer hydrocarbon groups exhibiting decreased polarity and increased non-polarity. The target veterinary drug residues are primarily polar substances with high polarity. Therefore, excessive extraction using highly polar solvents is avoided at the critical stage of the invention to minimize losses. According to the principle of like dissolves like, non-polar organic solvents interact with the hydrophobic long chains of fatty acids and neutral lipids through hydrophobic interactions (van der Waals forces), thereby dissolving these categories of lipids. n-Hexane is the most widely used lipid removal solvent, and although it is highly flammable, it is an effective extraction solvent for non-polar / neutral lipids while reducing matrix effects. Therefore, an acetonitrile-n-hexane saturated acetonitrile liquid-liquid extraction step was added to the pork matrix to remove a large amount of lipids with satisfactory recovery rates.

[0145] 2. Optimization of centrifugal temperature:

[0146] The samples were centrifuged at room temperature (20 ± 2 °C), 4 °C and -4 °C. Figure 3As shown. Taking fish (grass carp) as an example, the difference in the recovery of new veterinary drug residues at 4°C and -4°C was negligible. The recoveries of anthranilic acid, antipyrine and loperamide at -4°C were slightly higher than those at 4°C, but the deviation did not exceed 2%. However, compared with the latter two, the recoveries of most compounds centrifuged at room temperature decreased slightly, among which ormeprim showed a significant decrease (<9%). The centrifuge generates a lot of heat when running at high speed, and the protein or fat in the centrifuged sample may undergo qualitative changes at high temperatures. In view of the small difference in recovery between 4°C and -4°C temperatures, the experimental decision was to perform low-temperature centrifugation at 4°C.

[0147] 3. Optimization of purification steps:

[0148] Selection of purification powder and dosage of C18:

[0149] The composition of animal food matrix is ​​complex, containing a variety of fats and phospholipids that are easily extracted with acetonitrile. However, direct entry into the chromatographic column may lead to adsorption and subsequent elution difficulties. Long-term use of the system may cause increased pressure, so impurities such as fats and phospholipids need to be removed before mass spectrometry analysis. QuEChERS, as a typical dispersed solid phase extraction (DSPE) technology, uses the interaction between the adsorbent filler and the interfering matrix to achieve the purpose of decontamination and purification. It has the advantages of simple operation, high recovery rate, accurate detection results, and a wide range of applications. The main purification materials used in QuEChERS technology include N-(n-propyl) ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB), anhydrous magnesium sulfate (MgSO4), etc. PSA is used to remove organic acids, pigments and metal ions, while C18 has a greater ability to remove non-polar substances and can effectively remove long-chain lipids such as fats and fat-soluble pigments. GCB is mainly used to adsorb substances such as pigments. A series of experiments were conducted with the aim of comparing the efficacy of different purification powder ratios in different proportions: 1) 80 mg C18 + 1000 mg MgSO4 + 80 mg PSA; 2) 80 mg C18 + 1000 mg MgSO4 + 40 mg GCB; 3) 80 mg C18 + 1000 mg MgSO4 + 80 mg PSA + 40 mg GCB; 4) 80 mg C18 + 1000 mg MgSO4, e.g. Figure 4As shown. The results show that the addition of PSA improved the recovery of some substances to a certain extent, but the overall recovery effect of the substances was not as obvious as combination 4. The addition of GCB significantly reduced the recovery of several substances, especially the effect on 3,6-diamino-10-methylacridinium and 3,6-acridinium diamine. Therefore, C18 and MgSO4 were selected as purification reagents and their dosages were optimized. The mixed standard with a concentration of 10 ng / ml was added to beef, pork, chicken, shrimp and fish under the same conditions to investigate the purification effect of 40 mg, 60 mg, 80 mg, 100 mg, 60 mg, 80 mg, 100 mg and 120 mg C18 on new veterinary drug residues. Six replicates were performed for each matrix, and the average recoveries of the ten matrices are shown in Figure 5 (a, b). The results show that when C18 was 100 mg, most targets showed excellent recoveries in ten representative matrices. In contrast, when the amount of C18 powder was 40 mg or less, the recovery was significantly lower. Taking beef matrix ( Figure 5a ), the data showed that the recoveries of pentylenetetrazol, methylephedrine hydrochloride, and phenacetin at 80 mg C18 were slightly higher than those at 100 mg C18 (no more than 4%). Loperamide showed the highest recovery at 60 mg C18, while ciclesonide showed the highest recovery at 120 mg C18. When C18 was set to 40 mg, the recoveries of most substances decreased, except for butaquinate and antipyrine.

[0150] Dosage of MgSO4:

[0151] The presence of water reduces the concentration rate. When extracting by the QuEChERS method, salt is usually added during the extraction to reduce the solubility of veterinary drugs in the aqueous phase by salting out, thereby increasing the content of veterinary drugs in the organic phase, promoting the separation of the aqueous phase and the organic phase, and reducing the amount of water in the organic phase. The most commonly used salts are MgSO4, Na2SO4, and NaCl. Na2SO4 easily releases a large amount of heat when it comes into contact with water. However, during the extraction process, veterinary residues should be kept as low as possible to minimize fat leaching. Sodium chloride can remove some water-soluble compounds. Since MgSO4 is a more effective desiccant than Na2SO4 in removing residual water from organic solvents. After comparing various dehydrating salts, MgSO4 was finally selected as the dehydrating salt for this study, and its content was optimized ( Figure 6 ). When the concentration of MgSO4 was 1100 mg, 85% acetonitrile water was used as the extraction solvent to achieve the maximum extraction efficiency, and at the same time, the water was quickly removed, effectively reducing the nitrogen blowing time. Therefore, the optimal dosage of the purification step was determined to be 100 mg C18 and 1100 mg MgSO4.

[0152] Embodiment 6:

[0153] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. The homogenized sample is extracted and purified, including the following steps:

[0154] 3.1) For samples of cattle, sheep, horses, chicken, fish, shrimp, liver, milk and fat:

[0155] 3.1.1) Accurately weigh 2.0 g of sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells (the fat sample is heated in a 60°C water bath until it melts);

[0156] 3.1.2) After step 3.1.1), add 1 g NaCl to the milk sample, and add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the remaining samples, and shake and extract for 5 min;

[0157] 3.1.3) After step 3.1.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature (20 ± 2°C) or 4°C or -4°C;

[0158] 3.1.4) The supernatant obtained in step 3.1.3) was added to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortexed for 2 min, and centrifuged at 10000 r / min and 4°C for 5 min;

[0159] 3.1.5) After step 3.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow with nitrogen at 45°C until it is completely dry;

[0160] 3.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis;

[0161] 3.2) For pig samples:

[0162] 3.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells;

[0163] 3.2.2) After step 3.2.1), add acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the pig muscle sample and extract with shaking for 5 minutes;

[0164] 3.2.3) After step 3.2.2), the sample was extracted in an ultrasonic water bath for 15 min and centrifuged at 10,000 rpm for 5 min at room temperature (20 ± 2 °C) or 4 °C or -4 °C;

[0165] 3.2.4) The supernatant obtained in step 3.2.3) was mixed with 5 ml of acetonitrile-saturated n-hexane, vortexed for 2 min, and then centrifuged at 10,000 rpm at 4°C for 5 min;

[0166] 3.2.5) After step 3.2.4), transfer the acetonitrile layer to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortex for 2 min, and centrifuge at 10000 r / min at 4°C for 5 min;

[0167] 3.2.6) After step 3.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45°C;

[0168] 3.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis.

[0169] Embodiment 7:

[0170] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. The homogenized sample is extracted and purified, including the following steps:

[0171] 3.1) For samples of cattle, sheep, horses, chicken, fish, shrimp, liver, milk and fat:

[0172] 3.1.1) Accurately weigh 2.0 g of sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells (the fat sample is heated in a 60°C water bath until it melts);

[0173] 3.1.2) After step 3.1.1), add 1 g NaCl to the milk sample and 10 mL 85% acetonitrile to the remaining samples and extract by shaking for 5 min;

[0174] 3.1.3) After step 3.1.2), the sample was extracted in an ultrasonic water bath for 15 min and centrifuged at 10,000 rpm at 4°C for 5 min;

[0175] 3.1.4) Add the supernatant obtained in step 3.1.3) into a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex mix for 2 min, and centrifuge at 10000 rpm and 4°C for 5 min;

[0176] 3.1.5) After step 3.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow with nitrogen at 45°C until it is completely dry;

[0177] 3.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis;

[0178] 3.2) For pig samples:

[0179] 3.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min to allow the solution to fully penetrate into the tissue cells;

[0180] 3.2.2) After step 3.2.1), add 10 mL of 85% acetonitrile to the pig muscle sample and extract with shaking for 5 min;

[0181] 3.2.3) After step 3.2.2), the sample was extracted in an ultrasonic water bath for 15 min and centrifuged at 10,000 rpm at 4°C for 5 min;

[0182] 3.2.4) The supernatant obtained in step 3.2.3) was mixed with 5 ml of acetonitrile-saturated n-hexane, vortexed for 2 min, and then centrifuged at 10,000 rpm at 4°C for 5 min;

[0183] 3.2.5) After step 3.2.4), transfer the acetonitrile layer to a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex for 2 min, and centrifuge at 10000 rpm at 4°C for 5 min;

[0184] 3.2.6) After step 3.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45°C;

[0185] 3.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis.

[0186] Embodiment 8:

[0187] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. When performing HPLC-MS / MS analysis,

[0188] The chromatographic conditions are:

[0189] The chromatographic columns ACQUITY UPLC HSS T3 or ACQUITY UPLC BEH C18 were used for separation; the mobile phase A was methanol or acetonitrile, and the mobile phase B was 0.1% HCOOH, 0.2% HCOOH, 5mmol / L HCOONH4, 5mmol / L CH3COONH4 or 5mmol / L CH3COONH4+0.1%HCOOH; the elution conditions of the veterinary drug residues are: 5% mobile phase A, 0-2min; 5-50% mobile phase A, 2-2.5min; 50-85% mobile phase A, 2.5-6min; 85-90% mobile phase A, 2.5-6min; 90% mobile phase A at 9-10min; 90-95% mobile phase A at 10-10.1min, 95% mobile phase A at 10.1-12min, 95-5% mobile phase A at 12-12.5min, 5% mobile phase A at 12.5min-15min, and the total duration of the experiment is 15min. In addition, the flow rate is 0.25mL / min and the injection volume is 3.0μL;

[0190] Testing conditions:

[0191] Mass spectrometry was performed using a triple quadrupole mass spectrometer in electrospray positive ionization mode with:

[0192] The electrospray capillary voltage was 3.00 kV, and the ESI+cone voltage was 30 V; the dissolved gas flow rate was 1000 L / h, the collision gas flow rate was set to 0.15 mL / min, and the temperature was 200-500 °C. Qualitative analysis of each compound was performed under the multiple reaction monitoring (MRM) mode.

[0193] Embodiment 9:

[0194] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method described in the present invention, the following setting is particularly adopted: a chromatographic column ACQUITYUPLC HSS T3 is used, methanol is used as the mobile phase A, and 0.2% HCOOH is used as the mobile phase B; the triple quadrupole mass spectrometer uses a Waters Quattro Premier triple quadrupole mass spectrometer.

[0195] In order to achieve better results, the detection conditions were optimized in series:

[0196] When the analyte is transferred from the solution to the gas phase by ESI, three main processes are experienced. First, charged droplets are generated from the high-pressure capillary tip into which the analytical solution is injected. Second, the solvent evaporates from the charged droplets, and the charged droplets are reduced in size, thereby generating charged analytes. Finally, gas-phase charged (+ / -) analyte ions are formed. In the research and development stage, the present invention selects a single standard solution (100 ng / mL) to determine the precursor ions of the analyte in positive and negative ion modes. According to the optimized detector voltage conditions, the two fragment ions with the highest response are selected as the respective product ions. In positive ion electrospray ionization (ESI+) mass spectrometry, the most common ion adducts are [M+H]+, [M+NH4]+, [M+Na]+, [M+K]+[2M+H]+, [2M+NH4]+, [2M+Na]+. Ionized acidic residues (sulfonylamino, carboxyl, phenolic hydroxyl, etc.) easily form ion pairs with positive ions such as Na+ and NH4+. The results showed that most of the new veterinary drug residues formed [M+H]+ adducts (Table 2), with higher responses in positive ion mode. The optimal mass spectrometry parameters for each target veterinary drug (new veterinary drug residue) are shown in Table 2.

[0197] Table 2 Retention times and optimal operating mass spectrometry parameters for target veterinary drug analysis

[0198]

[0199]

[0200] * is the quantitative ion.

[0201] In order to achieve better results, the chromatographic column was optimized:

[0202] The separation of 19 new veterinary drug residues by ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm, Waters, USA) and ACQUITY UPLC HSS T3 (2.1 mm * 100 mm, 1.8 μm, Waters, USA) columns was compared. Although the ACQUITY UPLC BEH C18 column is commonly used as an ultra-high performance column for the analysis of a variety of analytes, it was found that the separation of new veterinary drug residues was not ideal. Most new veterinary drug residues are highly polar. The results showed that under the same liquid phase conditions, the HSS T3 column was used to separate new veterinary drug residues with satisfactory results, and had better peak shape and response than the BEH C18 column. The ACQUITY UPLC HSS T3 stationary phase is a C18 stationary phase compatible with 100% aqueous mobile phases and is more suitable for the retention of water-soluble and polar small organic compounds. Therefore, ACQUITY UPLC HSS T3 column was selected as the analytical column for the experiment.

[0203] In order to achieve better results, the mobile phase was optimized accordingly:

[0204] Generally speaking, polar solvents (such as water, methanol, acetonitrile, etc.) are used in ESI-MS experiments because they are prone to electrochemical reactions in the nozzle. The present invention compared the peak elution effects of methanol and acetonitrile in the research and development stage, and the elution ability of the methanol / water mobile phase was lower than that of the acetonitrile / water mobile phase. Thereby extending the retention time of the new veterinary drug residues, which is beneficial to the separation of chromatographic peaks. After comprehensive evaluation, methanol was selected as the organic phase (mobile phase A). With 0.1% HCOOH, 0.2% HCOOH, 5mmol / L HCOONH4, 5mmol / L CH3COONH4, and 5mmol / L CH3COONH4+0.1% HCOOH as the aqueous phase (mobile phase B), the separation effects and response differences of different aqueous phases on each target were investigated. The results show that the addition of HCOOH can effectively improve the peak shape and protonation ability, thereby promoting the formation of the [M+H]+ peak and enhancing the response. Compared with 0.1% HCOOH, 0.2% HCOOH has a higher response value and good peak shape, so methanol-0.2% formic acid water was finally selected as the mobile phase. Under the optimal conditions, 19 target veterinary drug residues were quickly and completely separated. The chromatogram is shown in Figure 1. Figure 1 shown.

[0205] Verification results:

[0206] 1. Matrix effect:

[0207] After sample extraction and cleanup, some common extracts may affect the ionization efficiency of target veterinary drug residues, resulting in enhanced or suppressed signal response of the target substance. This phenomenon is called matrix effect (ME). During the electrospray process, compounds may be affected by ionization and impurities, co-eluting analytes, or degradation products. These impurities, co-eluting analytes, or degradation products are generated during the process from the formation of charged droplets from the sample solution sprayed from the capillary tip to the charged ions entering the cone. Matrix compounds can change the physical properties of the droplets, including surface tension. There may also be competition between the matrix and analytes for the limited elemental charge on the droplets, which may interfere with the detection or recovery of the target substance. This interference manifests as reduced recovery or response. Matrix enhancement or suppression can also lead to false positive or false negative results.

[0208] Therefore, most researchers use a variety of techniques to minimize or eliminate the effects of the matrix, including optimizing sample preparation, adjusting chromatographic parameters, the use of internal standards, split flow settings, or the necessity of using standard additives. Therefore, the present invention conducted a preliminary study on the matrix effect of this method. The negative sample was extracted and cleaned up to obtain a blank matrix extraction solution. A mixed standard solution was prepared using the blank matrix extraction solution and methanol, and a matrix-matched calibration solution and a solvent standard solution were prepared respectively. These solutions were analyzed using the same method. ME was evaluated by the following equation:

[0209] Where A is the slope of the matrix standard curve and B represents the slope of the pure solvent standard curve.

[0210] Different substances have different matrix effects. ME<0 indicates matrix inhibition, and vice versa, it indicates matrix enhancement. The larger the absolute value, the stronger the matrix effect. When |ME|<20%, it is a weak matrix effect, indicating that the matrix effect is not significant; when |ME|<50%, it is a moderate matrix effect; when |ME|>50%, it is a strong matrix effect, which is considered significant. Typical matrices used in the present invention include poultry (chicken), livestock (beef, pork, sheep, horse), aquatic (grass carp, shrimp), liver, fat and milk matrices. These matrices were selected to evaluate the matrix effects of different product categories on the test targets. As shown in Table 3, the target showed varying degrees of matrix enhancement or matrix inhibition in all 10 matrices. New veterinary drug residues showed significant matrix inhibition in most matrices, and most of them also showed moderate or strong matrix effects, such as acetanilide, pentozolin, methylephedrine hydrochloride, phenacetin, medetomidine, yohimbine, and loperamide in shrimp showed strong matrix effects (|ME|>50%), and anthranilic acid, ormeprim and ciclesonide showed moderate matrix effects in pork (50%>|ME|>20%). In view of the inherent complexity of animal-derived food matrices, the present invention adopts matrix matching standard curve external standard method for quantification to reduce the influence of matrix effect on actual sample determination and improve the accuracy of the quantitative method.

[0211] Table 3 Matrix effects of target analytes in ten matrices

[0212]

[0213]

[0214]

[0215]

[0216] Method Validation:

[0217] A series of working standard solutions with concentrations of 0.01 ng / mL, 0.1 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL and 50.0 ng / mL were used. Matrix-matched calibration curves of 19 target veterinary drug residues were drawn, with the abscissa x representing the analyte concentration and the ordinate y representing the measured peak area. The results are shown in Table 4. The correlation coefficient (R2) of the working curve in the range of 0.01 to 50.0 ng / ml was 0.991 to 0.999, and the linear relationship was good. It is worth noting that for the same analyte, the detection limit (LOD) and quantification limit (LOQ) values ​​of different matrices were different. In cattle, the signal-to-noise ratios of the 19 new veterinary drug residues at the spike concentrations of 0.01 to 0.52 μg / kg were all greater than 3, meeting the LOD requirements, and the signal-to-noise ratios at the spike concentrations of 0.03 to 1.74 μg / kg were all greater than 10, meeting the LOQ requirements. The detection limits in chicken, shrimp, fish, pork, sheep, eggs, liver, fat and milk were 0.01-1.26μg / kg, 0.01-0.88μg / kg, 0.01-0.85μg / kg, 0.01-1.29μg / kg, 0.01-0.32, 0.01-0.69, 0.01-0.91, 0.01-0.37 and 0.01, respectively. -0.29 (S / N>3), and the limits of quantification were 0.04-4.20μg / kg, 0.03-2.94μg / kg, 0.04-2.84μg / kg, 0.03-4.31μg / kg, 0.02-1.07, 0.02-2.30, 0.02-3.05, 0.02-1.23 and 0.02-0.96 (S / N>10). By comparison, the limits of quantification of all analytes in this method are lower than the veterinary drug limit requirements stipulated in South Korea, Canada, the United States and the European Union. This shows that this method meets the requirements for routine detection of veterinary drug residues in multi-matrix animal-derived foods.

[0218] Table 4 Verification parameters and reference regulations for maximum residue limits of target veterinary drugs in ten samples

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] In addition, the recovery and relative standard deviation (RSD) were studied to evaluate the application accuracy and precision of the determination of 19 new veterinary drug residues. The method was validated according to the CODEX Guidelines (CAC / GL 71-2009) (Kim et al. 2021, CAC 2013) and the Guidelines for the Analysis of Residual Animal Drugs of the Ministry of Food and Drug Safety of Korea, with a recovery of 60-120% and a relative standard deviation of 20-30% or less as validation criteria for determining the recovery and RSD. The spiking level was determined according to the Applicability of MRLs for Veterinary Drugs in Foods, the requirements for the target substance limit in the Maximum Residue Limits (MRLs) of Veterinary Drugs in Foods, European Union Regulation (EU) No 43 / 2020, European Union Regulation (EC) No 470 / 2009, and Title 21 of the Code of Federal Regulations (US Food and Drug Administration 2023). The samples were spiked with 5, 10 and 50 μg / kg (0.6, 1.2 and 10 μg / kg for ciclesonide), and three quality control samples were analyzed six times on the same day and three consecutive days. The results showed that the target animal residues could achieve satisfactory recovery rates (60.3-118.5%) in all 10 matrices. The RSDs of intra-day and inter-day precision are shown in Table 5 (n=6), which are ≤15.6% and ≤19.8% in beef, ≤16.7% and ≤19.6% in pork, ≤17.4% and ≤20.2% in mutton, ≤18.4% and ≤20.4% in horse meat, ≤16.5% and ≤17.6% in fish, ≤17.2% and ≤18.7% in chicken, ≤16.5% and ≤19.8% in shrimp, ≤19.9% ​​and ≤20.6 in liver, ≤18.5% and ≤20.9% in fat, and ≤17.7% and ≤19.5% in milk. From the collected data, it can be seen that the developed UHPLC-MS / MS method has high precision and good accuracy.

[0228] Table 5 Average recoveries and RSDs of target veterinary drug residues at different spike levels, n = 6

[0229]

[0230]

[0231]

[0232] Comparison with other reporting methods:

[0233] The main parameters of the invention were compared with other LC-MS / MS methods reported in the literature for the detection of veterinary drug residues in animal-derived foods (Table 6). A study conducted by Kim et al. (2019) used LC-MS / MS to detect seven veterinary drug residues in milk. Five of them were found to be the same as the substances in the present invention. Similarly, Zhang et al. (2016b) used LC / ESI-MS / MS to detect four veterinary drug residues in pig muscle, milk and edible eggs. Three of the substances were the same as in this study. Nevertheless, the present invention was able to achieve lower limits of quantification and cover a wider range of matrices, including beef, chicken and fish, which makes it more adaptable. Zhang et al. (2016c) may be the first study to use LC-MS / MS detection technology for tetramisole, diethylcarbamazine and guaifenesin in animal-derived foods (milk, eggs and pig muscle). The gradient elution of the three target substances was completed within 35 min. When used, the present invention can complete the gradient elution of 19 veterinary drug residues in a shorter time (15 min), effectively shortening the detection time and reducing the amount of solvent used. Kim et al. (2021) used LC-MS / MS to identify 64 veterinary drug residues in flounder, eel and shrimp, including acetanilide, dl-methylephedrine, phenacetin, antipyrine, diethylcarbamazine, tripelennamine, yohimbine, loperamide, etc. In contrast, the present invention can achieve lower quantification limit detection requirements for multiple compounds when applied, including acetanilide, dl-methylephedrine hydrochloride, loperamide and diethylcarbamazine. In order to meet the regulatory restrictions set by South Korea, Canada, the European Union and the United States, a variety of different matrices were used for detection, and the results showed satisfactory accuracy and precision. Yohimbine is an α2-adrenergic receptor antagonist, which is most commonly used in veterinary medicine to reverse the effects of α2-receptor agonists xylazine and detomidine. However, the current focus of related detection methods is on plants, barks and dietary supplements, and there are few studies on the detection of this substance in animal-derived food matrices. Nakajima et al. (2011) developed an LC-ESI-MS / MS method for the determination of naquinate and buquinate in eight matrices (chicken muscle, liver, heart, etc.). In contrast, this method can detect a larger number of substances and involves a larger number of matrices. In a study published by Junge et al. (2022), an LC-MS / MS method was established for the determination of 115 veterinary drug residues in beef.Ten of them overlapped with those tested in this paper, but the LOQs were higher (acetanilide: 1.2 μg / kg, dl-methylephedrine: 1.0 μg / kg, phenacetin: 1.3 μg / kg, antipyrine: 1.7 μg / kg, diethylcarbamazine: 1.2 μg / kg, acriflavine: 2.7 μg / kg, tribenamine: 1.18 μg / kg, ormeprim: 1.6 μg / kg, yohimbine: 1.4 μg / kg, loperamide: 0.8 μg / kg) and did not adequately cover their matrix. The authors of the paper also showed that their LOQs were higher than those reported in previous beef multi-residue analysis studies. Therefore, the QuEChERS combined with UHPLC-MS / MS method proposed in this paper has more advantages.

[0234] Table 6 Comparison of this method with other LC-MS / MS detection methods

[0235]

[0236]

[0237]

[0238] Determination of pesticides in real samples:

[0239] The applicability of the optimized HPLC-MS / MS method was evaluated on authentic samples purchased from the local market. A total of 50 batches (5 batches per matrix) were analyzed and quantified using matrix-matched analytical calibration curves. The results showed that the above substances were not detected.

[0240] References:

[0241] Codex Alimentarius Commission(2013).CCRVDF Codex Committee on Residues of Veterinary Drugs in Foods(2012)Guidelines for the design and implementation of national regulatory food safety assurance program associated with the use of veterinary drugs in foodproducing animals(CAC / GL71-2009).

[0242] Fais,A.P.,Franco,R.S.B.,da Silva,A.F.B.,de Freitas,O.,&Paschoal,J.A.R.(2017).LC-MS / MS methods for sulfadimethoxine and ormetoprim analysis infeed and fish fillet and a leaching study for feed after alternativeprocedures for the incorporation ofdrugs.FoodAdditives&Contaminants:PartA,34(4),501-508。

[0243] Jung,Y.S.,Kim,D.-B.,Nam,T.G.,Seo,D.,&Yoo,M.(2022).Identification andquantification ofmulti-class veterinary drugs and their metabolites inbeefusing LC–MS / MS.Food chemistry,382,132313。

[0244] Kim,E.,Park,H.,Park,S.,Choi,J.,Yoon,H.J.,&Kim,J.-H.(2021).Simultaneous determination of multi-class veterinary drugs in fisheryproducts with liquid chromatography–tandem mass spectrometry.AppliedBiological Chemistry,64(1),40。

[0245] Kim,M.,Kim,N.,Kwon,H.,Ha,S.,Kim,H.,&Kim,J.(2019).Developmentof asimultaneous multi-residue analysis for screening and confirmation of7veterinary drugs in bovine milk by LC-MSMS.J.Prev.Vet.Med,43,68-73。

[0246] Nakajima,T.,Hayashi,H.,Sasamoto,T.,Kanda,M.,Kusano,T.,Matsushima,Y.,Kanai,S.,Takeba,K.,&Nagayama,T.(2011).Determination ofnequinateandbuquinolate in livestock products using liquid chromatography-tandemmassspectrometry.Shokuhin eiseigaku zasshi.Journal of the Food HygienicSociety ofJapan,52(3),178-182。

[0247] Park,J.A.,Abd El-Aty,A.,Zheng,W.,Kim,S.K.,Choi,J.m., A.,Shim,J.H.,&Shin,H.C.(2018).Determination ofmetoserpate,buquinolate,anddiclofenac in pork,eggs,and milk using liquid chromatography–tandem massspectrometry.Biomedical Chromatography,32(6),e4215。

[0248] Park,J.A.,Zhang,D.,Kim,D.S.,Kim,S.K.,Cho,K.S.,Jeong,D.,Shim,J.H.,Kim,J.S.,Abd El-Aty,A.,&Shin,H.C.(2015).Single-step multiresiduedeterminationoften multiclass veterinary drugs in pork,milk,and eggs usingliquidchromatography with tandem mass spectrometry.Journal of SeparationScience,38(16),2772-2780。

[0249] US Food and Drug Administration(2023).CFR-Code of FederalRegulationsTitle 21,www.accessdata.fda.gov / scripts / cdrh / cfdocs / cfCFR / CFRSearch.cfm?CFRPart=556&showFR=1.(Accessed 11 May 2024)。

[0250] Zhang,D.,Park,J.-A.,Kim,S.-K.,Cho,S.-H.,Jeong,D.,Cho,S.-M.,Yi,H.,Shim,J.-H.,Kim,J.-S.,&Abd El-Aty,A.(2016a).Simultaneous detectionofflumethasone,dl-methylephedrine,and 2-hydroxy-4,6-dimethylpyrimidine inporcine muscle and pasteurized cow milk using liquid chromatography coupledwith triple-quadrupole mass spectrometry.Journal ofChromatographyB,1012,8-16。

[0251] Zhang,D.,Park,Z.-Y.,Park,J.-A.,Kim,S.-K.,Jeong,D.,Cho,S.-H.,Shim,J.-H.,Kim,J.-S.,Abd El-Aty,A.,&Shin,H.-C.(2016b).A combined liquidchromatography-triple-quadrupole mass spectrometry method for the residualdetection of veterinary drugs in porcine muscle,milk,and eggs.Environmentalmonitoring and assessment,188,1-16。

[0252] Zhang,D.,Park,J.-A.,Kim,D.-S.,Kim,S.-K.,Shin,S.-J.,Shim,J.-H.,Shin,SC,Kim,J.-S.,Abd El-Aty,A.,&Shin,H.-C.(2016c).A simple extraction method for the simultaneous detection of tetramisole and diethylcarbamazine in milk, eggs, and porcine muscle using gradient liquid chromatography–tandem massspectrometry. Food chemistry, 192, 299-305.

[0253] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Determination of 19 new veterinary drug residues in animal-derived foods based on modified QuEChERS-HPLC-MS / MS method. Features: The steps include: 1) Preparation of standard stock solutions: Use the standard samples to prepare standard stock solutions of different mass concentrations, mixed standard working stock solutions with a mass concentration of 1 μg / mL, and working standard solutions of different mass concentrations; 2) Preparation of blank matrix standard curve: Take an appropriate amount of mixed standard working stock solution and add it to the blank sample residue after extraction and purification to prepare a series of matrix-matched mixed standard solutions of different concentrations. After filtering, measure by HPLC-MS / MS, and then draw a blank matrix standard curve; 3) Sample preparation: Homogenize the cattle, pig, sheep, horse, chicken, fish, shrimp, liver, milk, and fat samples, extract and purify the homogenized samples, and then perform HPLC-MS / MS analysis.

2. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 1, Features: The step 1) includes the following specific steps: 1.1) Accurately weigh 0.01 g of ciclesonide standard and 0.01 g of butaquinate standard into a 10 mL volumetric flask, add acetonitrile and methanol to dissolve and dilute the two standards to the scale, prepare a single standard ciclesonide stock solution and a single standard butaquinate stock solution with a mass concentration of 1000 μg / mL, and store at -20°C in the dark; 1.2) Each standard stock solution of the remaining 17 liquid standards was prepared as follows: 1 mL of the standard solution to be prepared was accurately transferred to a 10 mL volumetric flask, and methanol or acetonitrile was added to reach a final volume of 10 mL. Then, a standard stock solution with a mass concentration of 10 μg / mL or 100 μg / mL was prepared and stored at -20°C in a dark environment for use; 1.3) Accurately weigh appropriate amounts of 19 standard stock solutions, dilute with methanol, and prepare a mixed standard working stock solution with a mass concentration of 1 μg / mL; 1.4) Prepare a series of working standard solutions with concentrations of 0.01 ng / mL, 0.1 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL and 50.0 ng / mL.

3. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 1, Features: The step 2) is specifically as follows: 2.1) Take an appropriate amount of the mixed standard working stock solution and add it to the residue of 8 blank samples after extraction and purification, and blow dry it with nitrogen at 55℃; 2.2) Add 1 mL of methanol, vortex to dissolve, and prepare a matrix-matched series of mixed standard solutions with concentrations of 0.01, 0.1, 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 ng / mL, respectively. Pass through a 0.22 μm filter membrane and apply to the machine for HPLC-MS / MS determination; 2.3) Draw a blank matrix standard curve with the measured characteristic ion peak area as the ordinate and the corresponding standard solution concentration as the abscissa.

4. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 1, Features: The preparation method of the blank sample comprises: 2.1.1) For blank samples of cattle, sheep, horse, chicken, fish, shrimp, liver, milk and fat: 2.1.1.1) Accurately weigh 2.0 g of a single sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min; 2.1.1.2) After step 2.1.1.1), add 1 g NaCl to the milk sample and add acetonitrile, ethyl acetate, 0.1% formic acid in acetonitrile or 1% ammonia in acetonitrile to the remaining samples and extract by shaking for 5 min; 2.1.1.3) After step 2.1.1.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature or 4°C or -4°C; 2.1.1.4) Take the supernatant obtained in step 2.1.1.3) and add it to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortex mix for 2 min, and centrifuge at 10000 r / min and 4°C for 5 min; 2.1.1.5) After step 2.1.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow it to dryness at 45 °C nitrogen; 2.1.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain single-class blank samples of cattle, sheep, horse, chicken, fish, shrimp, milk, liver and fat; 2.1.2) For pig blank samples: 2.1.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min; 2.1.2.2) After step 2.1.2.1), add acetonitrile, ethyl acetate, 0.1% formic acid in acetonitrile or 1% ammonia in acetonitrile to the pig muscle sample and extract by shaking for 5 min; 2.1.2.3) After step 2.1.2.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature or 4°C or -4°C; 2.1.2.4) Mix the supernatant obtained in step 2.1.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 r / min at 4°C for 5 min; 2.1.2.5) After step 2.1.2.4), transfer the acetonitrile layer to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortex for 2 min, and centrifuge at 10000 r / min and 4℃ for 5 min; 2.1.2.6) After step 2.1.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45 °C; 2.1.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain a pig muscle blank sample.

5. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 4, Features: The preparation method of the blank sample comprises: 2.1.1) For blank samples of cattle, sheep, horse, chicken, fish, shrimp, milk, liver and fat: 2.1.1.1) Accurately weigh 2.0 g of a single sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min; 2.1.1.2) After step 2.1.1.1), add 1 g NaCl to the milk sample and 10 mL 85% acetonitrile to the remaining samples and extract by shaking for 5 min; 2.1.1.3) After step 2.1.1.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm at 4°C for 5 min; 2.1.1.4) Add the supernatant obtained in step 2.1.1.3) into a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex mix for 2 min, and centrifuge at 10000 rpm and 4°C for 5 min; 2.1.1.5) After step 2.1.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow it to dryness at 45 °C nitrogen; 2.1.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain single-class blank samples of cattle, sheep, horse, chicken, fish, shrimp, milk, liver and fat; 2.1.2) For pig blank samples: 2.1.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min; 2.1.2.2) After step 2.1.2.1), add 10 mL of 85% acetonitrile to the pig muscle sample and extract with shaking for 5 min; 2.1.2.3) After step 2.1.2.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm at 4°C for 5 min; 2.1.2.4) Mix the supernatant obtained in step 2.1.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 r / min at 4°C for 5 min; 2.1.2.5) After step 2.1.2.4), transfer the acetonitrile layer to a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex for 2 min, and centrifuge at 10000 r / min and 4°C for 5 min; 2.1.2.6) After step 2.1.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45 °C; 2.1.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain a pig muscle blank sample.

6. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 1, Features: The homogenized sample is then subjected to extraction and cleanup, which includes the following steps: 3.1) For samples of cattle, sheep, horses, chicken, fish, shrimp, liver, milk and fat: 3.1.1) Accurately weigh 2.0 g of sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min; 3.1.2 After step 3.1.1), add 1 g NaCl to the milk sample and acetonitrile, ethyl acetate, 0.1% formic acid acetonitrile or 1% ammonia acetonitrile to the remaining samples and extract by shaking for 5 min; 3.1.3) After step 3.1.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature or 4°C or -4°C; 3.1.4) Take the supernatant obtained in step 3.1.3) and add it to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortex mix for 2 minutes, and refrigerate centrifuge at 10000r / min and 4℃ for 5 minutes; 3.1.5) After step 3.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow it to dryness at 45 °C nitrogen; 3.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis; 3.2) For pig samples: 3.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min; 3.2.2 After step 3.2.1), add acetonitrile, ethyl acetate, 0.1% formic acid in acetonitrile or 1% ammonia in acetonitrile to the pig muscle sample and extract by shaking for 5 minutes; 3.2.3) After step 3.2.2), extract the sample in an ultrasonic water bath for 15 min and centrifuge at 10,000 rpm for 5 min at room temperature or 4°C or -4°C; 3.2.4) Mix the supernatant obtained in step 3.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 rpm at 4°C for 5 min; 3.2.5) After step 3.2.4), transfer the acetonitrile layer to a purification tube containing 40-120 mg of N-(n-propyl)ethylenediamine (PSA), octadecyl bonded silica gel (C18), graphitized carbon black (GCB) or anhydrous magnesium sulfate (MgSO4) and 1100 mg of MgSO4, Na2SO4 or NaCl, vortex for 2 min, and centrifuge at 10000 r / min and 4℃ for 5 min; 3.2.6) After step 3.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45 °C; 3.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis.

7. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 6, Features: The homogenized sample is then subjected to extraction and cleanup, which includes the following steps: 3.1) For samples of cattle, sheep, horses, chicken, fish, shrimp, liver, milk and fat: 3.1.1) Accurately weigh 2.0 g of sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the sample, and let it stand at room temperature for 30 min; 3.1.2) After step 3.1.1), add 1 g NaCl to the milk sample and 10 mL 85% acetonitrile to the remaining samples and extract by shaking for 5 min; 3.1.3) After step 3.1.2), the sample was extracted in an ultrasonic water bath for 15 min and centrifuged at 10,000 rpm at 4°C for 5 min; 3.1.4) Add the supernatant obtained in step 3.1.3) into a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex mix for 2 min, and centrifuge at 10000 rpm and 4°C for 5 min; 3.1.5) After step 3.1.4), transfer the supernatant to a 15 mL glass nitrogen blowpipe and blow it to dryness at 45 °C nitrogen; 3.1.6) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis; 3.2) For pig samples: 3.2.1) Accurately weigh 2.0 g of porcine muscle sample into a 50 mL screw-cap centrifuge tube, add an appropriate amount of mixed standard working stock solution to the porcine muscle sample, and let it stand at room temperature for 30 min; 3.2.2) After step 3.2.1), add 10 mL of 85% acetonitrile to the pig muscle sample and extract with shaking for 5 min; 3.2.3) After step 3.2.2), the sample was extracted in an ultrasonic water bath for 15 min and centrifuged at 10,000 rpm at 4°C for 5 min; 3.2.4) Mix the supernatant obtained in step 3.2.3) with 5 ml of acetonitrile-saturated n-hexane, vortex for 2 min, and then centrifuge at 10,000 rpm at 4°C for 5 min; 3.2.5) After step 3.2.4), transfer the acetonitrile layer to a purification tube containing 100 mg C18 and 1100 mg MgSO4, vortex for 2 min, and centrifuge at 10000 r / min and 4°C for 5 min; 3.2.6) After step 3.2.5), transfer the supernatant into a 15 mL glass nitrogen blowpipe and blow it to dryness with nitrogen at 45 °C; 3.2.7) Then add 1 mL of methanol to dissolve, vortex to dissolve the residue, and filter through a 0.22 μm microporous filter membrane to obtain the product required for HPLC-MS / MS analysis.

8. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 1, Features: When conducting HPLC-MS / MS analysis, The chromatographic conditions are: The separation was performed using ACQUITY UPLC HSS T3 or ACQUITY UPLC BEH C18 columns; mobile phase A was methanol or acetonitrile, and mobile phase B was 0.1% HCOOH, 0.2% HCOOH, 5 mmol / L HCOONH 4 、5 mmol / L CH3COONH 4 or 5 mmol / L CH3COONH 4 + 0.1% HCOOH; the elution conditions for the veterinary drug residues were: 5% mobile phase A, 0-2 min; 5-50% mobile phase A, 2-2.5 min; 50-85% mobile phase A, 2.5-6 min; 85-90% mobile phase A, 2.5-6 min; 90% mobile phase A at 9-10 min; 90-95% mobile phase A at 10-10.1 min, 95% mobile phase A at 10.1-12 min, 95-5% mobile phase A at 12-12.5 min, 5% mobile phase A at 12.5 min-15 min, and the total duration of the experiment was 15 min; in addition, the flow rate was 0.25 mL / min and the injection volume was 3.0 μL; Testing conditions: Mass spectrometry was performed using a triple quadrupole mass spectrometer in electrospray positive ionization mode with: The electrospray capillary voltage was 3.00 kV, and the ESI+ cone voltage was 30 V. The dissolved gas flow rate was 1000 L / h, the collision gas flow rate was set to 0.15 mL / min, and the temperature was 200-500 °C. Qualitative analysis of each compound was performed in the multiple reaction monitoring mode.

9. The method for determining 19 new veterinary drug residues in animal-derived foods based on the modified QuEChERS-HPLC-MS / MS method according to claim 8, Features: The chromatographic column ACQUITY UPLC HSS T3 was used for separation, the mobile phase A was methanol, and the mobile phase B was 0.2% HCOOH; the triple quadrupole mass spectrometer was Waters Quattro Premier triple quadrupole mass spectrometer.

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