Simultaneous determination of cross-border related pesticides and veterinary drugs residues in animal-derived foods by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Abstract

The application relates to the technical field of food safety detection, and discloses a method for synchronously quantitatively determining cross-border related pesticide and veterinary drug residues in animal-derived food by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, focuses on pesticide and veterinary drugs with different residue limits among main trade partners, aims to establish a high-efficiency and accurate high-throughput screening method, provide reliable technical support for cross-border food multi-target residue analysis, strengthen the management and control of international supervision difference residues, guarantee the safety of trade animal-derived products, and help the smooth development of international food trade; the method comprises the following steps: 1) standard solution preparation: single standard stock solution, mixed standard intermediate solution, blank matrix solution and series of matrix matching standard working solution are prepared respectively; 2) sample pretreatment: after the homogenized sample is weighed, vortexed, ultrasonically extracted and purified and filtered, a prepared sample is obtained; 3) UHPLC-QTOF-MS analysis is carried out on the prepared sample.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically, to a method for the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Background Technology

[0002] In recent years, pesticide and veterinary drug residues in animal-derived foods have become a global food safety hotspot, posing a threat to public health. Driven by the continued growth in global demand for livestock, poultry, and aquatic products, widespread pesticide contamination in feed and the irrational use of veterinary drugs in the farming process have significantly increased the risk of human exposure. Pesticides can enter animals through contaminated feed, environmental water bodies, or through cross-contamination, and their lipophilic residues tend to accumulate in edible tissues such as muscle and fat. Simultaneously, the irrational use of veterinary drugs also leads to excessive residues in animal-derived foods. Once residue levels exceed safety thresholds, they may cause chronic health hazards such as allergic reactions, antibiotic resistance, and potential carcinogenicity.

[0003] To this end, countries around the world have established residue monitoring systems and set maximum residue limits (MRLs) to protect public health. However, there are significant differences between the MRLs and controlled compound lists among major trading partners. Japan implemented a positive list system in 2006, initially covering 799 pesticide residue limits, and its regulatory system has been continuously updated and improved. South Korea has recently increased the number of controlled pesticides in livestock and aquatic products from 84 to 142, and veterinary drugs from 167 to 212. The US Department of Agriculture's regulatory system covers more than 425 pesticide and veterinary drug maximum residue limits in major exported agricultural products and animal-derived foods. The European Union established a unified pesticide residue regulatory framework through Regulation (EC) No 396 / 2005 and continues to revise it. As of 2025, its appendices cover 239 active pesticide ingredients, corresponding to more than 48,000 maximum residue limits. Meanwhile, regulations such as (EU) 2024 / 2633 and (EU) 2025 / 1164 strictly control pharmacologically active substances in animal-derived foods. Canada's Ministry of Health is responsible for publishing residue limits, and its official database includes 342 pesticides. In contrast, my country's updated standards GB2763-2026 and GB 31650-2019 have further improved residue management, covering more than 130 pesticide residues and 283 types / species of veterinary drugs related to animal-derived foods. However, significant differences remain with major trading partners in terms of the list of monitored substances and regulatory limits. These inconsistencies pose challenges to cross-border food safety supervision and urgently require targeted high-throughput analysis methods. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. This method focuses on pesticides and veterinary drugs for which residue limits differ among major trading partners, aiming to establish an efficient and accurate high-throughput screening method. This will provide reliable technical support for multi-target residue analysis in cross-border foods, strengthen the control of residues with international regulatory differences, ensure the safety of traded animal-derived products, and facilitate the smooth conduct of international food trade.

[0005] This invention is achieved through the following technical solution: a method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, comprising the following steps: 1) Preparation of standard solutions: Prepare the required single standard stock solution, mixed standard intermediate solution, blank matrix solution and a series of matrix-matched standard working solutions respectively; 2) Sample pretreatment: Weigh the homogenized sample and extract it by vortexing, ultrasonic extraction, purification and filtration to obtain the pre-prepared sample; 3) Perform UHPLC-QTOF-MS analysis on the samples.

[0006] To further improve the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: Step 1) includes the following specific steps: 1.1) Accurately weigh 1 mg ± 0.0001 g or 10 mg ± 0.0001 g of solid reference standard, place it in a 10 mL volumetric flask, dissolve it in acetonitrile or methanol or a mixture of acetonitrile and methanol, and prepare single standard stock solutions of 100.0 μg / mL and 1000.0 μg / mL respectively; 1.2) The 104 solid reference standards were divided into four categories: herbicides, insecticides / acaricides, fungicides, and veterinary drugs (Table 1). An appropriate amount of each category was mixed with the single standard stock solution and diluted with methanol to prepare a 5.0 μg / mL mixed standard intermediate solution. 1.3) Prepare a blank sample to obtain a blank matrix solution; 1.4) Dilute the mixed standard intermediate solution stepwise with blank matrix solution to prepare a series of matrix-matched standard working solutions of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 50.0, 100.0, and 200.0 ng / mL.

[0007] To further improve the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: The preparation of the blank sample includes the following steps: 1.3.1) Accurately weigh 5.00±0.02g of homogenized sample, place it in a 50mL centrifuge tube with a cap, add 2.0mL of water, vortex for 30s, and let stand for 10min; 1.3.2) Add 5.0 mL of acetonitrile / methanol (9:1, v / v) mixture containing 1% acetic acid, vortex for 2 min, and sonicate at 20 °C for 15 min; 1.3.3) Add the extraction salt packet, vortex vigorously for 1 min, and centrifuge at 4℃ and 10000 r / min for 10 min; 1.3.4) Transfer 2.0 mL of supernatant to a 15 mL centrifuge tube containing dispersive solid phase extraction (d-SPE) adsorbent, vortex for 1 min, and centrifuge at 4 °C and 10000 r / min for 5 min; after purification, filter the supernatant through a syringe filter to obtain a blank sample for later use.

[0008] To further improve the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: In step 1.3.2), the volume ratio (v / v) of acetonitrile to methanol in the acetonitrile / methanol mixture is 9:1; In step 1.3.3), the salt extraction package contains 2g magnesium sulfate, 0.5g sodium chloride, 0.5g trisodium citrate, and 0.25g disodium hydrogen citrate. In step 1.3.4), the dispersed solid-phase extraction (d-SPE) adsorbent is 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate, or 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent (MAX), or 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent (MAX) + 2.5 mg graphitized carbon black (GCB), or 50 mg magnesium sulfate + 12.5 mg octadecylsilane (C18) + 15 mg hydrophilic-lipophilic balanced polymer (HLB-P) + 10 mg mixed-mode cation exchange adsorbent (MCX) + 12.5 mg MgO. mg mixed-mode anion exchange adsorbent (MAX); the needle filter is a 0.22μm hydrophilic polyether sulfone (PES) needle filter.

[0009] To further improve the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: In step 1.3.4), the dispersive solid phase extraction (d-SPE) adsorbent is 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate.

[0010] To further improve the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: Step 2) includes the following specific steps: 2.1) Accurately weigh 5.00±0.02g of homogenized sample, place it in a 50mL capped centrifuge tube, add 2.0mL of water, vortex for 30s, and let stand for 10min; 2.2) Add 5.0 mL of acetonitrile / methanol (9:1, v / v) mixture containing 1% acetic acid, vortex for 2 min, and sonicate at 20 °C for 15 min; 2.3) Add the extraction salt packet, vortex vigorously for 1 min, and centrifuge at 4℃ and 10000 r / min for 10 min; 2.4) Transfer 2.0 mL of supernatant to a 15 mL centrifuge tube containing dispersive solid phase extraction (d-SPE) adsorbent, vortex for 1 min, and centrifuge at 4 °C and 10000 r / min for 5 min; after purification, filter the supernatant through a 0.22 μm needle filter and inject it into a sample vial.

[0011] To further improve the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following configuration is specifically adopted: In step 2.2), the volume ratio of acetonitrile to methanol in the acetonitrile / methanol mixture is 9:1; In step 2.3), the salt extraction package contains 2g magnesium sulfate, 0.5g sodium chloride, 0.5g trisodium citrate, and 0.25g disodium hydrogen citrate. In step 2.4), the dispersed solid-phase extraction (d-SPE) adsorbent consists of 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate, or 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent (MAX), or 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent (MAX) + 2.5 mg graphitized carbon black (GCB), or 50 mg magnesium sulfate + 12.5 mg octadecylsilane (C18) + 15 mg hydrophilic-lipophilic balanced polymer (HLB-P) + 10 mg mixed-mode cation exchange adsorbent (MCX) + 12.5 mg MgO. mg mixed-mode anion exchange adsorbent (MAX); the needle filter is a 0.22μm hydrophilic polyether sulfone (PES) needle filter.

[0012] To further improve the implementation of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods, the following setup is adopted: In step 2.4), the dispersive solid phase extraction (d-SPE) adsorbent consists of 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate.

[0013] To further improve the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), the following setup is specifically adopted: When performing UHPLC-QTOF-MS analysis on the samples, a 1290-6550 UHPLC-iFunnel Q-TOF-MS system is used; and during the analysis: The chromatographic separation conditions are as follows: An HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters) was used at a column temperature of 35 °C. Injection volumes were 1.0–5.0 μL in positive ion mode and 1.0–5.0 μL in negative ion mode. Mobile phase A was a 2 mmol / L ammonium formate aqueous solution containing 0.01% formic acid, and mobile phase B was a 2 mmol / L ammonium formate methanol solution containing 0.01% formic acid. The flow rate was 0.3 mL / min, and the gradient elution program was as follows: 2% mobile phase B 0–1.0 min, 2%–15% mobile phase B 1.0–1.5 min, 15%–40% mobile phase B 1.5–2.5 min, 40%–70% mobile phase B 2.5–18.0 min, 70%–98% mobile phase B 18.0–25.0 min, 98% mobile phase B 25.0–27.0 min, and 98%–2% mobile phase B… 27.0~27.1 min, 2% mobile phase B 27.1~30.0 min; The mass spectrometry analysis conditions were as follows: a QTOF 5600 mass spectrometer equipped with dual AJS ESI ion sources, operating simultaneously in positive and negative electrospray ionization modes; key ion source parameters: gas temperature 290℃, drying gas flow rate 11.0 L / min, nebulizer gas pressure 35 psig, sheath gas temperature 350℃, sheath gas flow rate 11.0 L / min, capillary voltage (VCap) 3500V, nozzle voltage 500V; fragmentation voltage 130V, skimmer voltage 65V, octet 1 RF voltage 750V; the mass-to-charge ratio (m / z) range for both full-scan MS and targeted MS / MS modes was 50~1000; manual tuning and calibration were performed using the instrument's built-in tuning function to ensure high quality and accuracy; during the analysis, reference standard solutions were continuously injected through the built-in reference mass calibration function to achieve real-time mass correction and stable mass accuracy throughout the analysis.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention focuses on pesticides and veterinary drugs for which residue limits differ among major trading partners, aiming to establish an efficient and accurate high-throughput screening method. Unlike traditional liquid chromatography-tandem mass spectrometry (LC-MS / MS) and gas chromatography-tandem mass spectrometry (GC-MS / MS), high-resolution mass spectrometry (HRMS) employs a full-scan mode, which can acquire complete compound information and avoid missed detections, giving it a significant advantage in high-throughput multi-target screening.

[0015] This invention establishes a modified QuEChERS pretreatment method combined with a UHPLC-QTOF-MS high-throughput method to achieve rapid and accurate detection (including pesticide and veterinary drug screening and quantification) of 104 novel pesticide and veterinary drug residues in animal-derived foods. This method provides reliable technical support for multi-target residue analysis in cross-border food products, strengthens the control of residues with international regulatory differences, ensures the safety of traded animal-derived products, and facilitates the smooth conduct of international food trade.

[0016] This invention is simple to operate, widely applicable, and highly sensitive, with accuracy, precision, and linearity all meeting requirements. The optimized QuEChERS scheme efficiently removes matrix interference, minimizes non-specific adsorption, and alleviates matrix inhibition, ensuring reliable qualitative and quantitative results.

[0017] The detection limit and quantitation limit of this invention are far lower than the maximum residue limits set by the EU, Japan, South Korea, the US, and Canada, and fully meet the requirements for cross-border trade monitoring (except for Cyclosone). Detailed Implementation

[0018] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0020] To achieve better technical results, the instruments, materials, and reagents involved in this invention include: The following equipment was used to prepare ultrapure water: an Agilent Technologies 1290-6550 UHPLC-iFunnel Q-TOF-MS system with a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1×100 mm); a Guangdong Jiemeng Ultrasonic Industry Co., Ltd. SN-30D-40 LCD single-frequency ultrasonic cleaner; a Mettler Toledo XS205 electronic analytical balance (Grevenssee, Switzerland); a Sigma Laboratory Centrifuge 3-18KS benchtop refrigerated centrifuge (Osterrod, Harzberg, Germany); an IKA Group MS3 vortex mixer and a T25 digital laboratory homogenizer (Staufen, Germany); and an Edgeson ultrapure water system (with a biological filter) from Shanghai Edgeson Instruments Co., Ltd.

[0021] The 104 pesticide and veterinary drug analytical standards (i.e., solid reference standards) were mainly purchased from Shanghai Anpu Experimental Technology Co., Ltd., Tianjin Alta Technology Co., Ltd., Tianjin Tanmo Quality Inspection Technology Co., Ltd., Beijing Manhag Biotechnology Co., Ltd., Guangzhou Jiatu Technology Co., Ltd., and Shanghai Aladdin Biochemical Technology Co., Ltd. Detailed information such as compound molecular formula, CAS number, solvent, and pesticide category is shown in Table 1.

[0022] Acetonitrile and methanol of liquid chromatography-mass spectrometry grade were purchased from Thermo Fisher Scientific (Ilkisch, France); formic acid of high performance liquid chromatography grade was purchased from Chengdu Kelong Chemical Reagent Factory (Sichuan); glacial acetic acid of high performance liquid chromatography grade was purchased from Chengdu Kelong Chemical Co., Ltd. (Sichuan); ammonium formate of high performance liquid chromatography grade (≥99.0%) was purchased from Shanghai Ron Chemical Technology Co., Ltd. The extraction salt pack (2g magnesium sulfate / 0.5g sodium chloride / 0.5g trisodium citrate / 0.25g disodium hydrogen citrate) was purchased from Shanghai Qingyun Chemical Technology Co., Ltd. The dispersive solid-phase extraction (d-SPE) adsorbent combinations are as follows, all purchased from Shanghai Qingyun Chemical Technology Co., Ltd.: 25mg ethylenediamine-N-propylsilane (PSA) + 25mg octadecylsilane (C18) + 75mg magnesium sulfate; 25mg ethylenediamine-N-propylsilane (PSA) + 25mg octadecylsilane (C18) + 75mg magnesium sulfate + 10mg mixed-mode anion exchange adsorbent (MAX); 25mg ethylenediamine-N-propylsilane (PSA) + 25mg octadecylsilane (C18) + 75mg magnesium sulfate + 10mg mixed-mode anion exchange adsorbent (MAX); mg mixed-mode anion exchange adsorbent (MAX) + 2.5 mg graphitized carbon black (GCB); 50 mg magnesium sulfate + 12.5 mg octadecylsilane (C18) + 15 mg hydrophilic-lipophilic balanced polymer (HLB-P) + 10 mg mixed-mode cation exchange adsorbent (MCX) + 12.5 mg mixed-mode anion exchange adsorbent (MAX).

[0023] Waters Corporation (Milford, Massachusetts, USA) Oasis PRIME HLB solid phase extraction column (3cc / 60mg) and Agilent Technologies (Santa Clara, California, USA) Captiva EMR-Lipid solid phase extraction column (3mL / 300mg).

[0024] Table 1. Basic Information on 104 Pesticide and Veterinary Drug Analytical Standards (i.e., Solid Reference Standards)

[0025] Note: Abbreviations: QOSI = extraquinone inhibitor; SDHI = succinate dehydrogenase inhibitor; QOI = extraquinone inhibitor; DMI = demethylation inhibitor; CAA = carboxylamide; OSBPI = oxosterol-binding protein inhibitor; CNS = central nervous system.

[0026] Data processing was performed using Agilent MassHunter PCDL Manager software. All target standards were analyzed under established UHPLC-QTOF-MS conditions to construct a Personal Compound Database and Spectral Library (PCDL). The PCDL comprises two core components: a primary precise mass database (precise mass of the parent ion, molecular formula, ionization mode, retention time, chemical structure, isotope distribution, and abundance) and a secondary mass spectrometry library containing fragment ions and their corresponding collision energies (CE). MS and MS / MS data were acquired using Agilent MassHunter Workstation Data Acquisition (version B.05.01), and data processing was performed using Agilent MassHunter qualitative analysis (versions B.05.01 / 10.0) and quantitative analysis (version B.06.00) software.

[0027] The clear identification criteria for compounds are as follows: 1. Retention time deviation within ±0.1 min; 2. Precursor ion mass error within ±5 ppm; 3. MS¹ library matching score >80%, MS² library matching score >70%. Compounds that meet all the identification criteria and are not detected in the process blank are considered positively detected in the sample. All tables were created using Microsoft Office Excel.

[0028] Example 1: Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QFS-MS) is used for the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods. This method focuses on pesticides and veterinary drugs with differing residue limits among major trading partners, aiming to establish an efficient and accurate high-throughput screening method. It provides reliable technical support for multi-target residue analysis in cross-border foods, strengthens the control of residues with international regulatory differences, ensures the safety of traded animal-derived products, and facilitates the smooth conduct of international food trade. The method includes the following steps: 1) Preparation of standard solutions: Prepare the required single standard stock solution, mixed standard intermediate solution, blank matrix solution and a series of matrix-matched standard working solutions respectively; 2) Sample pretreatment: Weigh the homogenized sample and extract it by vortexing, ultrasonic extraction, purification and filtration to obtain the pre-prepared sample; 3) Perform UHPLC-QTOF-MS analysis on the samples.

[0029] Example 2: This embodiment is a further optimization based on the above embodiment. The parts identical to the aforementioned technical solutions will not be repeated here. Furthermore, to better realize the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: Step 1) includes the following specific steps: 1.1) Preparation of single standard stock solution: Accurately weigh 1 mg ± 0.0001 g or 10 mg ± 0.0001 g of solid reference standard, place it in a 10 mL volumetric flask, and dissolve it in acetonitrile, methanol or acetonitrile / methanol mixture to prepare single standard stock solutions of 100.0 μg / mL and 1000.0 μg / mL respectively; 1.2) Preparation of mixed standard intermediate solution: 104 solid reference standards were divided into four categories: herbicides, insecticides / acaricides, fungicides and veterinary drugs (Table 1). An appropriate amount of each category was taken and mixed with the single standard stock solution, and diluted with methanol to prepare a 5.0 μg / mL mixed standard intermediate solution. 1.3) Preparation of blank matrix solution: Prepare a blank sample to obtain a blank matrix solution; 1.4) Preparation of a series of matrix-matched standard working solutions: The mixed standard intermediate solution was gradually diluted with blank matrix solution to prepare a series of matrix-matched standard working solutions with concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 50.0, 100.0, and 200.0 ng / mL.

[0030] Example 3: This embodiment is a further optimization based on any of the above embodiments. The parts identical to the aforementioned technical solutions will not be repeated here. Furthermore, to better realize the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: The preparation of blank samples and sample pretreatment both include the following steps: S1. Accurately weigh 5.00±0.02g of homogenized sample, place it in a 50mL centrifuge tube with a cap, add 2.0mL of water, vortex for 30s, and let stand for 10min. S2. Add 5.0 mL of acetonitrile / methanol (9:1, v / v) mixture containing 1% acetic acid, vortex for 2 min, and sonicate at 20℃ for 15 min. S3. Add the extraction salt packet, vortex vigorously for 1 min, and centrifuge at 4℃ and 10000 r / min for 10 min; Furthermore, to better realize the present invention, the concentration step has been optimized: Nitrogen blowing concentration can enrich target analytes and lower the limits of detection and quantitation. This invention further investigates the effect of nitrogen blowing concentration on the recovery rate of target compounds. After purification, 1 mL of the supernatant was transferred to a 15 mL glass tube and evaporated with nitrogen at 40 °C until nearly dry. The solution was then redissolved in 0.5 mL of methanol-water (1:1, v / v) containing 0.1% formic acid. The effects of nitrogen blowing concentration and direct injection without concentration were compared at a spiking level of 50 μg / kg. The results showed that nitrogen blowing concentration adversely affected the recovery rate of some target compounds (pentanetetrazole, rivanol, acetanilide, and guaiacol), possibly due to thermal decomposition or volatilization loss of these compounds during nitrogen blowing evaporation.

[0031] Although nitrogen blowing is a routine enrichment technique in sample pretreatment, it can still lead to the loss of some analytes in practical applications; especially for non-targeted analysis, where the types and physicochemical properties of unknown components are unpredictable, the losses caused by evaporation and concentration are more pronounced. Considering the research objectives and subsequent non-targeted screening requirements, nitrogen blowing concentration is not used in routine sample pretreatment (and in the preparation of blank samples) to retain target components and unknown compounds to the greatest extent possible, ensuring accurate and reliable results for both targeted and non-targeted analyses. The optimal concentration procedure is as follows: S4. Transfer 2.0 mL of supernatant to a 15 mL centrifuge tube containing dispersive solid phase extraction (d-SPE) adsorbent, vortex for 1 min, and centrifuge at 4 °C and 10000 r / min for 5 min; after purification, filter the supernatant through a syringe filter; for the preparation of blank samples, the blank sample is obtained at this time for later use; for sample pretreatment, the obtained sample is injected into a sample vial.

[0032] Example 4: This embodiment is a further optimization based on any of the above embodiments. The parts identical to the aforementioned technical solutions will not be repeated here. Furthermore, to better realize the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following configuration is specifically adopted: the dispersive solid-phase extraction (d-SPE) adsorbent is 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate (this is a preferred configuration) or 25 mg ethylenediamine-N-propylsilane (PSA) + 75 mg magnesium sulfate (C18). A) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent (MAX) or 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent (MAX) + 2.5 mg graphitized carbon black (GCB) or 50 mg magnesium sulfate + 12.5 mg octadecylsilane (C18) + 15 mg hydrophilic-lipophilic balanced polymer (HLB-P) + 10 mg mixed-mode cation exchange adsorbent (MCX) + 12.5 mg mixed-mode anion exchange adsorbent (MAX); the needle filter is a 0.22 μm hydrophilic polyethersulfone (PES) needle filter.

[0033] Example 5: This embodiment is a further optimization based on any of the above embodiments. The parts that are the same as those in the aforementioned technical solutions will not be repeated here. In order to better realize the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the extraction conditions, QuEChERS, etc. are optimized.

[0034] (a) Optimization of extraction conditions: Acetonitrile and methanol are commonly used extraction solvents for analyzing pesticide and veterinary drug residues in food. Acetonitrile has excellent solubility, wide applicability, and moderate polarity, enabling efficient extraction of most pesticides and veterinary drugs with fewer interfering matrix components. In contrast, methanol has better solubility for some polar or weakly basic compounds, but pure methanol easily forms hydrogen bonds with proteins and polysaccharides, leading to increased co-extraction impurities and interfering with separation and quantification. Therefore, a mixed solvent system with acetonitrile as the main component and a small amount of methanol can balance analyte solubility and matrix purity, making it more suitable for multi-residue analysis.

[0035] Studies have shown that adding appropriate amounts of acid modifiers can improve extraction efficiency: the acidic environment adjusts the pH of the system, prevents analyte degradation, promotes the protonation of alkaline pesticides and veterinary drugs, and improves their solubility and partition efficiency into the organic phase; at the same time, it disrupts the hydrogen bonds and hydrophobic interactions between the target analyte and matrix components such as proteins and polysaccharides, releasing bound analytes and improving recovery rates. Water is usually added before extraction to fully disperse the sample, improve solvent wettability, wet the dry and dense matrix, increase the contact area, promote the release of the target analyte, and simultaneously reduce matrix viscosity and homogenization resistance, improving extraction repeatability.

[0036] This invention uses an acetonitrile / methanol (9:1, v / v) mixture as the main extraction solvent, systematically compares four extraction systems, and screens for optimal conditions. Using blank samples of beef (representing livestock and poultry meat matrix) and fish (representing aquatic product matrix) as matrices, a mixed standard intermediate solution is added to achieve a final concentration of 50 μg / kg for each compound. 5.0 g of homogenized sample is accurately weighed, 2.0 mL of water or 0.5% formic acid aqueous solution is added, followed by 5.0 mL of the corresponding extraction solvent and extraction salt mixture, and comparative extraction experiments are conducted.

[0037] The results showed that the recovery rates of the formic acid-containing extraction system were unsatisfactory, with significant differences between beef and fish matrices. A few compounds showed a sharp drop in recovery (approaching zero) in beef, while this phenomenon was more pronounced in the fish matrix. Typical examples include dichloroquinoline, indoxofensulfuron, pyraclostrobin, and fluthiazopyrone. Formic acid, as a small-molecule organic acid, can cause severe denaturation and structural modification of proteins in the fish matrix, inducing conformational changes or intermolecular cross-linking and aggregation. Target analytes are physically encapsulated in protein precipitates, making extraction difficult and significantly reducing recovery rates. Simultaneously, some compounds in the formic acid-containing system exhibited artificially high apparent recoveries (>120%), occurring in both matrices but more prominently in the fish matrix, such as trifluralin, tetrazolium, chlorpyrifos, and pyrazosulfuron-methyl. It is speculated that formic acid, acting as a matrix modifier, significantly enhances the detection response of target analytes, leading to an overestimation of recovery rates. In contrast, the water-1% acetic acid (acetonitrile / methanol = 9:1) system was stable in both matrices, with most compounds showing recoveries within the ideal range of 70%–120%. In addition, all experimental steps are preferably carried out under low temperature conditions; fat is the main interfering substance in animal-derived foods, and freezing centrifugation is used to change the physical state of fat in the extract, remove some fat from high-fat samples, and simplify the subsequent purification process.

[0038] Therefore, the preferred embodiment for the preparation of blank samples and sample pretreatment of the present invention is as follows: the volume ratio of acetonitrile to methanol in the acetonitrile / methanol mixture is 9:1; the extraction salt package contains 2g magnesium sulfate, 0.5g sodium chloride, 0.5g trisodium citrate, and 0.25g disodium hydrogen citrate. (II) QuEChERS optimization and comparison with solid phase extraction columns: In animal-derived foods, matrix components such as lipids, incompletely precipitated proteins, organic acids, and pigments are easily co-extracted into the organic phase with the target analyte (solid reference standard), causing a significant matrix effect that interferes with detection and affects the recovery rate of the target compound. QuEChERS, a widely used dispersive solid-phase extraction (d-SPE) technique for animal-derived food sample pretreatment, employs a variety of purification adsorbents, mainly including magnesium sulfate, C18, PSA, GCB, MAX, MCX, and HLB-P. Among them, anhydrous magnesium sulfate mainly removes water from the extraction system; C18 efficiently removes non-polar co-extractants such as lipids; PSA removes sugars, organic acids, and some polar pigments from the matrix through weak anion exchange and hydrogen bonding interaction; GCB has strong adsorption for compounds containing planar aromatic rings (Ar-) and conjugated double bonds (-C=CC=C-); MAX is a mixed-mode anion exchange adsorbent that selectively removes acidic interfering substances such as carboxyl groups (-COOH) and phenolic hydroxyl groups (-OH); MCX is a strong cation exchange adsorbent suitable for targeted purification of alkaline compounds; and HLB-P is a hydrophilic-lipophilic balanced adsorbent with broad applicability to polar to moderately polar compounds.

[0039] Based on the aforementioned adsorbents, this invention systematically compared the recovery rates of six different purification schemes in beef and fish matrices. The results showed that Scheme 1 (25 mg ethylenediamine-N-propylsilane (PSA), 25 mg octadecylsilane (C18), and 75 mg anhydrous magnesium sulfate) exhibited the best recovery rates in both matrices, with most target compounds recovering within the acceptable range of 60%–120%. This indicates that the classic adsorbent combination has excellent broad-spectrum applicability to beef and fish matrices, effectively removing major interfering substances such as fatty acids and pigments, while avoiding excessive adsorption of target analytes. The recovery rates of some compounds (mainly acidic substances and planar aromatic compounds) in Scheme 2 (25 mg ethylenediamine-N-propylsilane (PSA), 25 mg octadecylsilane (C18), 75 mg anhydrous magnesium sulfate, 10 mg mixed-mode anion exchange adsorbent (MAX)) and Scheme 3 (25 mg ethylenediamine-N-propylsilane (PSA), 25 mg octadecylsilane (C18), 75 mg anhydrous magnesium sulfate, 10 mg mixed-mode anion exchange adsorbent (MAX) and 2.5 mg graphitized carbon black (GCB)) decreased significantly. This was mainly due to the non-specific adsorption of acidic target substances by MAX and the irreversible retention of planar aromatic compounds by GCB. Scheme 4 (50 mg anhydrous magnesium sulfate, 12.5 mg octadecylsilane (C18), 15 mg hydrophilic-lipophilic balanced polymer (HLB-P), 10 mg mixed-mode cation exchange adsorbent (MCX), and 12.5 mg mixed-mode anion exchange adsorbent (MAX)) employs a combination of multiple adsorbents in mixed modes, but the recovery rate fluctuates greatly, with some compounds exceeding the acceptable range. Specifically, acidic compounds containing -COOH or sulfonylurea groups (dichloroquinoline acid, imidazolinol, mesosulfuron-methyl) are adsorbed by MAX through anion exchange; basic compounds containing amine groups or basic heterocycles (tripyridine, yohimbine, rivanol) are retained by MCX through cation exchange; and polar compounds containing amides or hydroxyl groups (acetanilide, guaiacol) form hydrogen bonds with HLB-P. The results indicate that combinations of multiple adsorbents are prone to synergistic or antagonistic effects, making it difficult to achieve stable and efficient broad-spectrum purification; and Scheme 4 performs better overall than beef substrate in fish substrate.

[0040] Based on this, this invention further compares the purification and recovery efficiency of the QuEChERS method with two commercially available solid-phase extraction (SPE) columns specifically designed for animal-derived matrices (Scheme 5 (Oasis PRIME HLB (3cc / 60mg) solid-phase extraction cartridges) and Scheme 6 (Captiva EMR-Lipid (3mL / 300mg) cartridges) for the target pesticide residues. The extraction procedure for the SPE group is the same as that for QuEChERS. After centrifugation, 2mL of the supernatant is loaded onto the SPE column, and the eluent is collected for instrumental analysis. The results show that the extract purified by the commercially available SPE column is lighter in color and clearer, especially in the beef matrix where the solution color is significantly lighter; however, neither of the two straight-through SPE columns achieved satisfactory recovery rates in either matrix. The reason may be that after a large amount of lipids and proteins in the animal-derived matrix are adsorbed by the adsorbent, the exposed active sites on the surface may further bind hydrophobic and moderately polar target compounds through non-specific interactions and remain on the column, ultimately leading to a low recovery rate of the target analyte.

[0041] Therefore, the preferred method for preparing blank samples and pretreatment of samples in this invention is as follows: the dispersive solid phase extraction (d-SPE) adsorbent is 25 mg ethylenediamine-N-propylsilane (PSA) + 25 mg octadecylsilane (C18) + 75 mg magnesium sulfate.

[0042] Example 6: This embodiment is a further optimization based on any of the above embodiments. The parts identical to the aforementioned technical solutions will not be repeated here. Furthermore, to better realize the method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry as described in this invention, the following setup is specifically adopted: When performing UHPLC-QTOF-MS analysis on the samples, a UHPLC-iFunnel Q-TOF-MS system (model 1290-6550) was used; and during the analysis: The chromatographic separation conditions are as follows: An HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters) was used at a column temperature of 35 °C. Injection volumes were 1.0–5.0 μL in positive ion mode and 1.0–5.0 μL in negative ion mode. Mobile phase A was a 2 mmol / L ammonium formate aqueous solution containing 0.01% formic acid, and mobile phase B was a 2 mmol / L ammonium formate methanol solution containing 0.01% formic acid. The flow rate was 0.3 mL / min, and the gradient elution program was as follows: 2% mobile phase B 0–1.0 min, 2%–15% mobile phase B 1.0–1.5 min, 15%–40% mobile phase B 1.5–2.5 min, 40%–70% mobile phase B 2.5–18.0 min, 70%–98% mobile phase B 18.0–25.0 min, 98% mobile phase B 25.0–27.0 min, and 98%–2% mobile phase B… 27.0~27.1 min, 2% mobile phase B 27.1~30.0 min; The mass spectrometry analysis conditions were as follows: a QTOF 5600 mass spectrometer equipped with dual AJS ESI ion sources, operating simultaneously in positive and negative electrospray ionization modes; key ion source parameters: gas temperature 290℃, drying gas flow rate 11.0 L / min, nebulizer gas pressure 35 psig, sheath gas temperature 350℃, sheath gas flow rate 11.0 L / min, capillary voltage (VCap) 3500V, nozzle voltage 500V; fragmentation voltage 130V, skimmer voltage 65V, octet 1 RF voltage 750V; the mass-to-charge ratio (m / z) range for both full-scan MS and targeted MS / MS modes was 50~1000; manual tuning and calibration were performed using the instrument's built-in tuning function to ensure high quality and accuracy; during the analysis, reference standard solutions were continuously injected through the built-in reference mass calibration function to achieve real-time mass correction and stable mass accuracy throughout the analysis.

[0043] To improve UHPLC-QTOF-MS analysis, this invention optimizes the instrument conditions, primarily focusing on improvements in mass spectrometry, chromatographic conditions, and injection volume. (a) Optimization of mass spectrometry conditions: To achieve simultaneous detection of multiple pesticide and veterinary drug residues under switching between positive and negative ion modes, the selection of ionization mode needs to be combined with the chemical structural characteristics of the analytes and their protonation / deprotonation / adduct behavior in the electrospray ionization source. The target analytes (pesticide and veterinary drug analytical standards) of this invention cover compounds with significant polarity differences, such as benzimidazoles, sulfonylureas, triazoles, amides, macrolides, organophosphates, pyrethroids, and methoxyacrylates. Common functional groups in their structures include amino (-NH2), amide (-CONH-), nitrogen-containing heterocycles (pyridine, pyrimidine, imidazole rings, etc.), carbonyl (C=O), carboxyl (-COOH), sulfonyl (-SO2-), and phenolic hydroxyl (-OH).

[0044] In positive ion mode, amino groups have a strong affinity for protons in nitrogen-containing heterocyclic compounds and readily combine with H+ in acidic environments. + Formation [M+H] + Parent ion; it is worth noting that phenothiazine nitrogen-containing heterocyclic compounds ionize into radical molecular ions M in positive ion mode. + • Instead of the protonated molecular ion [M+H] commonly seen in mass spectrometry. + This unique ionization behavior is similar to that of medium- to long-chain tetrazine compounds, mainly forming M... + ·、[M+H] + The extremely low abundance confirms that some nitrogen-containing heterocyclic compounds, due to their unique structural characteristics, are more likely to form radical molecular ions in positive ion mode, providing a reference for the mass spectrometric characterization of this type of compound. Some compounds containing carbonyl groups or ester bonds readily react with NH4+. + [M+NH4] is formed. + Addition ions.

[0045] In negative ion mode, acidic groups such as carboxyl, phenolic hydroxyl, and sulfonyl groups are easily deprotonated to form [MH]. - Parent ions; compounds containing strongly electronegative elements such as fluorine and chlorine are also suitable for negative ion electrospray ionization, as these elements can promote the formation of deprotonated anions of the analyte.

[0046] Based on the above ionization mechanism, full-scan MS analysis was performed in both positive and negative ion modes. Of all analytes (pesticide and veterinary drug analytical standards), 3,6-acrididane, acetanilide, antipyrine, loperamide, naloxone, omepramine, triamcinolone, pentylenetetrazol, fluconazole, benomyl, fluthiazoline, cyclopyrimethanil, imidacloprid, flupyradifurone, pyrimethanil, imidacloprid, spinosad J, spinosad L, pyraclostrobin, and diflubenzuron only responded in positive ion mode; benzimidazole, baquipulin, clostridium, and dithiocarbamate were detected only in negative ion mode; the remaining compounds (pesticide and veterinary drug analytical standards) showed acceptable responses in both ionization modes. Ultimately, the mode with the higher signal intensity was selected for qualitative and quantitative analysis.

[0047] To obtain confirmatory structural information, collision energies (CE) were systematically optimized in MS / MS mode in increments of 5 eV, ranging from 5 to 50 eV. The optimal CE was determined based on the response behavior of each compound, and the corresponding product ion spectra were recorded. Detailed information on ionization modes, retention times (Rt), qualitative / quantitative ion pairs, and collision energies (CE) is shown in Table 2.

[0048] Table 2 Characteristic mass spectrometry data of the target analytes

[0049] Note: An asterisk (*) indicates that the isotope peak was selected as the parent ion for mass spectrometry / mass spectrometry (MS / MS) analysis.

[0050] (II) Optimization of chromatographic conditions: The mobile phase composition and gradient elution program significantly affect analyte ionization efficiency, chromatographic retention behavior, and matrix effects. Methanol, as a proton donor (α=0.98), is significantly superior to acetonitrile (α=0.19) as a proton solvent. Its solvation shell can stabilize the protonation sites of nitrogen-containing heterocyclic and amino compounds in positive ion mode through hydrogen bonding, thereby enhancing [M+H]. + Response. The addition of formic acid provides sufficient H+ for the positive ion mode. + Formic acid promotes the protonation of amino and heterocyclic nitrogen atoms. Studies have shown that adding formic acid to the mobile phase can significantly improve the signal intensity and detection coverage of various metabolites in positive ion mode. In negative ion mode, low concentrations of formic acid maintain a weakly acidic environment in the mobile phase (pH 3.5–4.0), which avoids excessive inhibition of deprotonation of acidic groups such as carboxyl groups, and effectively neutralizes the activity of silanol groups and reduces secondary interactions.

[0051] Ammonium formate also acts as a volatile buffer salt to maintain stable ionic strength and provide NH4+. + With HCOO - As an addition ligand, it significantly expands the detection range in both positive and negative ion modes. Although ammonium acetate exhibits superior signal intensity and retention time stability in negative ion mode, ammonium formate's advantage in maintaining pH stability during gradient elution is irreplaceable. In this invention, all target compounds (pesticide and veterinary drug analytical standards) achieved satisfactory peak shapes and signal responses in negative ion mode, fully demonstrating that ammonium formate is suitable for the analytical system established in this invention.

[0052] The organic and aqueous phases use the same additive composition (0.01% formic acid + 2 mmol / L ammonium formate) to ensure a constant background ion intensity during gradient elution, effectively avoiding fluctuations in ionization efficiency caused by changes in ion concentration. This is crucial for rapid switching between positive and negative ion modes. Based on a methanol-water gradient program optimized according to GB 23200.121, the initial high proportion of the aqueous phase (98%) promotes the early elution of strongly polar interfering substances (salts, sugars, organic acids) and reduces matrix inhibition; the high proportion of the organic phase at the end ensures complete elution of weakly polar analytes. Good chromatographic separation of 104 pesticides and veterinary drugs was achieved within 30 minutes.

[0053] (III) Optimization of injection volume: After confirming that the target compounds (analytical standards for pesticides and veterinary drugs) have good chromatographic separation performance, the effect of injection volume of 1-5 μL on peak shape and response was systematically investigated.

[0054] In positive ion mode, different compounds exhibit significant differences in their response to injection volume: Antipyrine, omeprine, and methyl sulfone, among others, show highly sensitive peak shapes to injection volume; at 1 μL, the peaks are sharp and symmetrical, while beyond 1 μL, the peaks broaden significantly, flatten at the peak tip, and tail, affecting accurate quantification. Compounds like phenacetin show even higher response and optimal peak shape at 2 μL; further increasing the injection volume leads to peak distortion. These phenomena stem from the space charge effect of the ion source or detector saturation. Excessive ion flux exceeds the linear dynamic range of the mass spectrometer, resulting in a non-linear relationship between peak area / peak height and analyte concentration, introducing negative quantitative bias. Therefore, a 1 μL injection volume is chosen in positive ion mode to balance peak shape quality and quantitative accuracy.

[0055] In negative ion mode, the target analyte response is generally weak. Increasing the injection volume from 1 μL to 5 μL results in a proportional increase in peak area and signal intensity, with no significant peak shape degradation. Therefore, a 5 μL injection volume is used in negative ion mode to improve detection sensitivity.

[0056] Therefore, for better analysis, the injection volume was 1.0 μL in positive ion mode and 5.0 μL in negative ion mode. Mobile phase A was a 2 mmol / L ammonium formate aqueous solution containing 0.01% formic acid, and mobile phase B was a 2 mmol / L ammonium formate methanol solution containing 0.01% formic acid, with a flow rate of 0.3 mL / min.

[0057] Mass spectrometry analysis conditions: Key ion source parameters: gas temperature 290℃, dry gas flow rate 11.0 L / min, nebulizer gas pressure 35 psig, sheath gas temperature 350℃, sheath gas flow rate 11.0 L / min, capillary voltage (VCap) 3500V, nozzle voltage 500V; fragmentation voltage 130V, skimmer voltage 65V, octet 1 RF voltage 750V.

[0058] Example 7: Based on the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method proposed in any of the above embodiments for the simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods, this invention also validates the method: Following the EU SANTE / 11312 / 2021 guideline, the specificity, sensitivity, accuracy, and precision of the method were validated. Specificity: The risk of false positives was assessed using blank matrix samples. Sensitivity: Evaluation was based on the limit of detection (SDL), limit of quantitation (LOQ), linearity, and coefficient of determination (R²). SDL is the lowest spiked concentration that can be detected in at least 95% of blank meat samples after multiple concentration levels and 20 replicates per level. LOQ is the lowest spiked concentration with a recovery rate of 70%–120% and a relative standard deviation (RSD) ≤20%. Standard curves were plotted with peak area as the dependent variable and calibration concentration as the independent variable. 104 curves were established within their respective linear ranges, with the lower limit of the linear range set as the SDL for each compound, to validate the linearity. Spiking test: Standard solution was added to the extract sample, mixed thoroughly, and allowed to stand overnight. Accuracy: Expressed as recovery rate in repeated assays spiked at concentrations of 1 to 10 times the corresponding maximum residue limit (LOQ); precision is expressed as RSD, including intra-day precision for repeated assays on the same day and inter-day precision for three consecutive days. Matrix effect (ME): Calculated using the formula ME (%) = (slope of matrix-matched curve / slope of solvent standard curve - 1) × 100%, and categorized as weak effect (|ME| ≤ 20%), moderate effect (20% < |ME| ≤ 50%), and strong effect (|ME| > 50%).

[0059] Specifically, method validation includes the following aspects: (1) Specificity: Two sample matrices, beef and fish, were selected. The modified QuEChERS method was used to purify the samples to obtain analytical solutions. These solutions were then serially diluted with a mixed standard intermediate solution to obtain spiked analytical solutions. UHPLC-QTOF-MS was used to analyze both the analytical solutions and the spiked analytical solutions to check for interfering peaks at retention times for each compound. The results showed that no interfering peaks were observed in any of the 104 compounds, indicating good method specificity.

[0060] (2) Evaluation of matrix effect: The inherent matrix effect of complex biological matrices such as beef and fish often leads to significant deviations when directly quantifying pesticide and veterinary drug residues using solvent standard curves, affecting analytical accuracy. Therefore, it is essential to assess the matrix effect and determine its impact on method precision and result accuracy. The matrix effect originates from the ionization competition between matrix impurities and the target analyte at the ion source, resulting in either enhanced or suppressed target compound signals.

[0061] As shown in Table 3, most pesticide and veterinary drug analytical standards exhibited significant matrix inhibition effects (ME < 0) in both matrices, with the inhibition being more pronounced in the fish matrix. In the beef matrix, nearly half of the compounds showed a weak matrix effect (|ME| < 20%), while about one-fifth (represented by propoxyimidazole, fensulfuron-methyl, chlorpyrifos, quinclorac, dithiophenol, dimethomorph, and sulfadiazine) showed a strong matrix effect (|ME| > 50%). In the fish matrix, most compounds showed moderate or strong matrix effects, with only 10 compounds—yohimbine, fluthiazoline, succinate, buquinazone, dimethomorph, pyraclostrobin, leprosylate A4, phenacetin, flonicamid, and clostridium—showing a weak matrix effect. Based on these results, this invention employs a matrix-matched standard curve external standard method for quantification, reducing the interference of matrix effects on the determination of actual samples and improving the accuracy and reliability of the analytical results.

[0062] (3) Limit of detection and limit of quantitation: The limits of detection (SDL) and quantitation (LOQ) were determined by serially diluting the standard solutions with blank matrix extracts. SDL was the lowest spiked concentration that could be detected in at least 95% of 20 replicates; LOQ was the lowest concentration with a recovery rate of 70%–120% and an RSD ≤ 20%. In exceptional cases, average recoveries exceeding 70%–120% but with consistent results (RSD ≤ 20%) were acceptable. The 104 target compounds showed good linearity in beef and fish matrices within the range of 0.2–200 ng / mL (R² > 0.9913), with SDL of 0.200–5.00 ng / mL (0.200–5.00 μg / kg) and LOQ of 1.00–20.0 ng / mL (1.00–20.0 μg / kg) (Table 3).

[0063] Table 3. Linear regression equations, correlation coefficients (R²), matrix effects, limits of detection (SDLs), and limits of quantitation (LOQs) for target compounds in beef and fish matrices.

[0064] This method can simultaneously perform qualitative and quantitative analysis of 104 pesticide and veterinary drug residues, with limits of quantification (LOQs) far below the maximum residue limits (MRLs) set by the EU, Japan, South Korea, the US, and Canada. The only exception is cyclosunide, which, while failing to meet the EU's stringent 0.6 μg / kg MRL, fully complies with Japan's positive list system's default limit of 0.01 mg / kg and the regulations of the Korean Ministry of Food and Drug Safety (MFDS). Most other regions do not have specific MRLs for cyclosunide, and this compound requires further optimization to achieve even lower detection levels. Overall, this method is reliable, economical, and suitable for routine monitoring of pesticide and veterinary drug residues in animal-derived foods.

[0065] (4) Accuracy and precision: Beef and fish blank matrix samples were selected, and three spiking levels—low (LOQ), medium (2–5 times LOQ / MRL), and high (5–10 times LOQ / MRL)—were set within the linear range, with six replicates per level to evaluate the accuracy and precision of the method. After spiking the mixed standard solutions, the samples were allowed to stand overnight to ensure sufficient absorption of the spike residue by the matrix. The average recoveries of 104 target compounds in the two matrices ranged from 60.7% to 118.9%, with relative standard deviations (RSDs) ranging from 2.1% to 19.7%. Overall, the established method met the accuracy and precision requirements.

[0066] (5) Residual analysis of actual samples: To evaluate the practicality of the established method, 53 domestic and imported samples (17 beef, 15 fish, 12 chicken, and 9 shrimp) collected from the Chinese market were analyzed using this method. The results showed that only one sample of domestic fish contained fluoramide (2.34 μg / kg). Previous studies have reported the presence of fluoramide residues in various freshwater environments, with the highest detected concentration exceeding 1400 μg / L. It is speculated that the fluoramide residues in fish stem from the fish's long-term exposure to polluted water environments, leading to the bioaccumulation of fluoramide in fish tissues. No residues of the target compound were detected in the remaining samples.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, characterized by: Includes the following steps: 1) Preparation of standard solutions: Prepare the required single standard stock solution, mixed standard intermediate solution, blank matrix solution and a series of matrix-matched standard working solutions respectively; 2) Sample pretreatment: Weigh the homogenized sample and extract it by vortexing, ultrasonic extraction, purification and filtration to obtain the pre-prepared sample; 3) Perform UHPLC-QTOF-MS analysis on the samples.

2. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to claim 1, characterized in that: Step 1) includes the following specific steps: 1.1) Weigh 1 mg ± 0.0001 g or 10 mg ± 0.0001 g of solid reference standard, place it in a 10 mL volumetric flask, and dissolve it in acetonitrile, methanol or a mixture of acetonitrile and methanol to prepare single standard stock solutions of 100.0 μg / mL and 1000.0 μg / mL respectively; 1.2) The 104 solid reference standards were divided into four categories: herbicides, insecticides / acaricides, fungicides, and veterinary drugs. An appropriate amount of each category was mixed with the single standard stock solution and diluted with methanol to prepare a 5.0 μg / mL mixed standard intermediate solution. 1.3) Prepare a blank sample to obtain a blank matrix solution; 1.4) Dilute the mixed standard intermediate solution stepwise with blank matrix solution to prepare a series of matrix-matched standard working solutions of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 50.0, 100.0, and 200.0 ng / mL.

3. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to claim 2, characterized in that: The preparation of the blank sample includes the following steps: 1.3.1) Weigh 5.00±0.02g of homogenized sample, place it in a 50mL capped centrifuge tube, add 2.0mL of water, vortex for 30s, and let stand for 10min; 1.3.2) Add 5.0 mL of acetonitrile / methanol mixture containing 1% acetic acid, vortex for 2 min, and sonicate at 20℃ for 15 min; 1.3.3) Add the extraction salt packet, vortex vigorously for 1 min, and centrifuge at 4℃ and 10000 r / min for 10 min; 1.3.4) Transfer 2.0 mL of supernatant to a 15 mL centrifuge tube containing dispersed solid-phase extraction adsorbent, vortex for 1 min, and centrifuge at 4 °C and 10000 r / min for 5 min; after purification, filter the supernatant through a syringe filter to obtain a blank sample for later use.

4. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to claim 3, characterized in that: In step 1.3.2), the volume ratio of acetonitrile to methanol in the acetonitrile / methanol mixture is 9:

1. In step 1.3.3), the salt extraction package contains 2g magnesium sulfate, 0.5g sodium chloride, 0.5g trisodium citrate, and 0.25g disodium hydrogen citrate. In step 1.3.4), the dispersed solid-phase extraction adsorbent is 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate, or 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent, or 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent + 2.5 mg graphitized carbon black, or 50 mg magnesium sulfate + 12.5 mg octadecylsilane + 15 mg hydrophilic-lipophilic balanced polymer + 10 mg mixed-mode cation exchange adsorbent + 12.5 mg mixed-mode anion exchange adsorbent; the needle filter is a 0.22 μm hydrophilic polyethersulfone needle filter.

5. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to claim 4, characterized in that: In step 1.3.4), the dispersed solid-phase extraction adsorbent is 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate.

6. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to any one of claims 1 to 5, characterized in that: Step 2) includes the following specific steps: 2.1) Weigh 5.00±0.02g of homogenized sample, place it in a 50mL centrifuge tube with a cap, add 2.0mL of water, vortex for 30s, and let stand for 10min; 2.2) Add 5.0 mL of acetonitrile / methanol mixture containing 1% acetic acid, vortex for 2 min, and sonicate at 20℃ for 15 min; 2.3) Add the extraction salt packet, vortex vigorously for 1 min, and centrifuge at 4℃ and 10000 r / min for 10 min; 2.4) Transfer 2.0 mL of supernatant to a 15 mL centrifuge tube containing dispersed solid-phase extraction adsorbent, vortex for 1 min, and centrifuge at 4 °C and 10000 r / min for 5 min; after purification, filter the supernatant through a syringe filter and inject it into a sample bottle.

7. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to claim 6, characterized in that: In step 2.2), the volume ratio of acetonitrile to methanol in the acetonitrile / methanol mixture is 9:

1. In step 2.3), the salt extraction package contains 2g magnesium sulfate, 0.5g sodium chloride, 0.5g trisodium citrate, and 0.25g disodium hydrogen citrate. In step 2.4), the dispersed solid-phase extraction adsorbent consists of 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate, or 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent, or 25 mg ethylenediamine-N-propylsilane + 25 mg octadecylsilane + 75 mg magnesium sulfate + 10 mg mixed-mode anion exchange adsorbent + 2.5 mg graphitized carbon black, or 50 mg magnesium sulfate + 12.5 mg octadecylsilane + 15 mg hydrophilic-lipophilic balanced polymer + 10 mg mixed-mode cation exchange adsorbent + 12.5 mg mixed-mode anion exchange adsorbent; the needle filter is a 0.22 μm hydrophilic polyethersulfone needle filter.

8. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to claim 7, characterized in that: In step 2.4), the dispersed solid-phase extraction adsorbent consists of 25 mg ethylenediamine-N-propylsilane, 25 mg octadecylsilane, and 75 mg magnesium sulfate.

9. The method for simultaneous quantitative determination of cross-border related pesticide and veterinary drug residues in animal-derived foods using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry according to any one of claims 1-5 and 7-8, characterized in that: When performing UHPLC-QTOF-MS analysis on the samples, a UHPLC-iFunnel Q-TOF-MS system (model 1290-6550) was used; and during the analysis: The chromatographic separation conditions are as follows: An HSS T3 column was used at a column temperature of 35℃. Injection volumes were 1.0–5.0 μL in positive ion mode and 1.0–5.0 μL in negative ion mode. Mobile phase A was a 2 mmol / L ammonium formate aqueous solution containing 0.01% formic acid, and mobile phase B was a 2 mmol / L ammonium formate methanol solution containing 0.01% formic acid. The flow rate was 0.3 mL / min. The gradient elution program was as follows: 2% mobile phase B 0–1.0 min, 2%–15% mobile phase B 1.0–1.5 min, 15%–40% mobile phase B 1.5–2.5 min, 40%–70% mobile phase B 2.5–18.0 min, 70%–98% mobile phase B 18.0–25.0 min, 98% mobile phase B 25.0–27.0 min, 98%–2% mobile phase B 27.0–27.1 min, and 2% mobile phase B… 27.1~30.0 min; The mass spectrometry analysis conditions were as follows: QTOF 5600 mass spectrometer, equipped with dual AJS ESI ion sources, operating simultaneously in positive and negative electrospray ionization modes; key ion source parameters: gas temperature 290℃, dry gas flow rate 11.0 L / min, nebulizer gas pressure 35 psig, sheath gas temperature 350℃, sheath gas flow rate 11.0 L / min, capillary voltage (VCap) 3500V, nozzle voltage 500V; fragmentation voltage 130V, skimmer voltage 65V, octet 1 RF voltage 750V; the mass-to-charge ratio range for both full-scan MS and targeted MS / MS modes was 50–1000.

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