Solid-phase extraction process method for detecting veterinary drug residues in animal-derived sample

Abstract

The invention discloses a solid-phase extraction process method for detecting veterinary drug residues in an animal-derived sample, and belongs to the technical field of analysis of harmful residues in food. The method comprises the steps of sample pretreatment, solid-phase extraction column activation balance, sample loading, multi-stage washing and core collaborative elution. Especially, core collaborative elution adopts a collaborative elution method combining three-stage pH gradient elution and two-time competitive acetone pulse injection. The method comprises the following steps: firstly, eluting sulfonamides and quinolones under an acidic condition; then injecting acetone to competitively remove phospholipid; then, the tetracycline and macrolide drugs are eluted under the neutral condition; injecting acetone again for deep purification; and finally, eluting the chloramphenicol medicine under an alkaline condition. According to the method, the problem that the recovery rate and the purification degree are difficult to consider when multiple types of veterinary drug residues in the egg high-phospholipid complex matrix are synchronously detected is effectively solved, and the accuracy, the sensitivity and the reproducibility of subsequent LC-MS / MS analysis are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of harmful residue analysis technology in food, specifically to a solid-phase extraction process for detecting veterinary drug residues in animal-derived samples. Background Technology

[0002] As a widely consumed animal food, the issue of veterinary drug residues in eggs directly impacts consumer health and public health. To control poultry diseases, various antibiotics, including fluoroquinolones, sulfonamides, tetracyclines, and macrolides, are commonly used in poultry farming. Excessive residues of these drugs in eggs can induce drug resistance, trigger allergic reactions, or pose long-term carcinogenic risks. Therefore, Chinese regulations such as GB 31650-2019 and EU regulations (EU) No 37 / 2010 set strict limits for veterinary drug residues in eggs, requiring detection methods to possess high sensitivity, accuracy, and throughput.

[0003] Solid-phase extraction (SPE), a key step in sample pretreatment, is mainly used to enrich target analytes and remove interferences from complex matrices, providing pure samples for subsequent high-sensitivity analyses such as LC-MS / MS. This technique is widely used in veterinary drug residue detection due to its ease of operation, low solvent consumption, and high purification efficiency.

[0004] However, traditional SPE methods have significant limitations when handling highly complex matrices such as eggs. Eggs are rich in protein (approximately 12%), fat (approximately 10%), and phospholipids (1–2%). Phospholipids readily compete with veterinary drugs for adsorption or co-elution, interfering with detection. Different classes of veterinary drugs exhibit significant differences in pH (pKa) and polarity. For example, tylosin (pKa≈7.8) is more readily adsorbed under acidic conditions, sulfamethoxazole (pKa≈5.7) is more readily eluted under alkaline conditions, while amphoteric drugs such as enrofloxacin have strong adsorption at specific pH values. Traditional SPE methods often use a single pH elution buffer, making it difficult to achieve high recoveries for all target analytes when extracting multiple classes of veterinary drugs. Recovery rates for some drugs are below 60%, failing to meet the requirements for simultaneous detection of multiple residues.

[0005] Furthermore, phospholipids exhibit strong adsorption on reversed-phase adsorbents such as C18 and HLB, easily co-eluting with the target analyte and causing ion inhibition in LC-MS / MS, severely impacting the accuracy and sensitivity of quantification. Existing improved methods, such as mixed-mode SPE columns or linear pH gradient elution, while effective to some extent, still struggle to balance recovery and purification efficiency due to high cost, complex operation, or the introduction of more impurities in the intermediate pH range.

[0006] Therefore, based on the general SPE column, there is an urgent need to develop a new process that achieves efficient fractional elution and deep removal of phospholipids for multiple types of veterinary drugs through synergistic optimization of eluent composition, pH and operation sequence, so as to simultaneously achieve the detection goals of high recovery rate, high purification degree and high throughput. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a solid phase extraction process that can efficiently remove phospholipid interference and achieve simultaneous detection of high recovery rates for multiple types of veterinary drugs.

[0008] This invention is implemented as follows: A solid-phase extraction process for detecting veterinary drug residues in animal-derived samples includes the following steps: S1. Sample pretreatment: The homogenized animal-derived sample is mixed with acetonitrile, and protein precipitation, centrifugation and filtration are performed to obtain the sample loading solution. S2. Solid-phase extraction column activation and equilibration: A hydrophilic-lipophilic balanced polymer adsorbent solid-phase extraction column is used, which is activated by organic solvent and equilibrated by ultrapure water in sequence. S3. Sample loading: The sample solution is loaded onto the solid-phase extraction column after activation equilibrium. After the sample solution has completely flowed through the solid-phase extraction column, it is dried. S4. Multi-stage washing: The solid phase extraction column is washed in stages using washing solutions composed of at least two different pH values ​​and organic solvents; S5. Core Synergistic Elution: To achieve efficient separation of multiple types of veterinary drugs and deep purification of phospholipids, the following sub-steps are executed sequentially through a combination of three-stage pH gradient elution and two competitive acetone pulse injections: S5-1, First stage pH gradient elution: Elute using an eluent with a pH of 2.5-4.0 and containing 50%-80% organic solvent, and collect the first eluted fraction; S5-2, First competitive acetone pulse injection: Inject an acetone aqueous solution with a concentration of 60%-90% into the solid phase extraction column, and discard the effluent after standing. S5-3, Second stage pH gradient elution: Elute using an eluent with a pH of 6.5-8.0 and containing 40%-60% organic solvent, and collect the second eluted fraction; S5-4, Second competitive acetone pulse injection: Inject an acetone aqueous solution with a concentration of 70%-95% into the solid phase extraction column, and discard the effluent after standing; S5-5, Third-stage pH gradient elution: Elution is performed using an eluent with a pH of 8.5-10.0 and containing 80%-95% organic solvent, and the third eluted fraction is collected.

[0009] Further, in step S1, the volume-to-mass ratio of the acetonitrile to the animal-derived sample is 1:1 to 3:1.

[0010] Furthermore, in step S1, the centrifugation conditions are 4°C and 8000-12000 rpm for 5-10 minutes.

[0011] Further, in step S1, the filtration is performed using a 0.22 μm polytetrafluoroethylene filter membrane.

[0012] Further, in step S2, the organic solvent used for activation is methanol or acetonitrile; wherein the amount of the organic solvent added is 2.0-5.0 mL, and its flow rate is 0.5-2 mL / min.

[0013] Furthermore, in step S3, the flow rate of the sample solution being loaded is 0.5-2 mL / min.

[0014] Further, in step S4, the multi-stage washing includes: First washing stage: Use a washing solution with a pH of 3.0-5.0 and containing 1%-10% methanol for washing; Second washing: Use a washing solution with a pH of 6.0-8.0 and containing 10%-30% acetonitrile for washing.

[0015] Further, in step S5, the volume of the first and second competitive acetone pulse injections is independently 0.5-2 mL, the injection flow rate is 0.2-1 mL / min, and the residence time in the solid phase extraction column is 0.5-2 min. The organic solvent in the first, second, and third pH gradient eluents is acetonitrile; wherein the volume of the eluent injected is independently 0.5-2 mL, and the injection flow rate is 0.2-1 mL / min.

[0016] Furthermore, the animal-derived sample is an egg sample.

[0017] Furthermore, it also includes subsequent analytical steps: after concentrating and reconstituted the collected first, second and third elution components, qualitative and quantitative analysis is performed by liquid chromatography-tandem mass spectrometry; wherein, the third elution component is acidified by adding an acidic solution before concentration.

[0018] The present invention has the following advantages: (1) This invention provides an optimized elution environment for veterinary drugs with different physicochemical properties by using three-stage pH gradient elution for different acidity, alkalinity and polarity. The recovery rate of 10 representative veterinary drugs in five major categories, including sulfonamides, quinolones, tetracyclines, macrolides and chloramphenicol, reaches 78.8%-93.0%, which effectively avoids the problems of uneven recovery rate and low recovery rate of some drugs (such as tetracyclines) caused by traditional single elution mode (such as as low as 55.3% in Comparative Example 1).

[0019] (2) This invention utilizes two competitive acetone pulse injections to achieve efficient and selective removal (removal rate >96%) of phospholipids, the main interfering substance in eggs, by specifically competingly binding acetone molecules with phospholipids to adsorbent sites. This significantly weakens the ion inhibition effect caused by phospholipids in subsequent LC-MS / MS analysis, and the matrix effect is significantly improved from -20.1% to -41.2% in Comparative Example 1 to a weak level of -4.5% to -18.5%, greatly enhancing the accuracy and detection sensitivity of trace veterinary drug quantification.

[0020] (3) This invention, through segmented evaluation of phospholipid removal efficiency, confirms that each elution stage achieves high-efficiency purification: the first elution component has low phospholipid residue due to the acidic environment and pre-washing effect; the second and third components benefit from the deep purification of acetone pulse, with an overall phospholipid removal rate of >96%. At the same time, the alkaline elution component is immediately acidified, which effectively ensures the chemical stability of alkaline sensitive drugs such as chloramphenicol, and keeps the recovery rate of alkaline sensitive drugs such as chloramphenicol stable at over 87%.

[0021] (4) The core elution method of this invention produces a synergistic effect of "1+1>2". The acetone pulse effectively removes phospholipids during the elution interval, purifies the microenvironment of the solid phase extraction column, and creates a purer adsorption-desorption interface for subsequent pH gradient elution, thereby ensuring a high recovery rate for all types of veterinary drugs; while the precise fractional elution sequence avoids a large amount of co-elution of phospholipids and target substances, reducing the purification burden of the acetone pulse.

[0022] (5) This invention is based on a universal hydrophilic-lipophilic balance (HLB) polymer solid-phase extraction column, which does not rely on expensive or special packing materials, thus reducing detection costs. This method is easy to operate and provides an efficient, reliable and green pretreatment solution for high-throughput and accurate monitoring of complex and multi-category veterinary drug residues in animal-derived samples (especially eggs). Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] This invention selects 10 representative veterinary drugs from five major classes as target analytes, including sulfonamides (sulfadimidine, sulfamethoxazole), quinolones (ciprofloxacin, enrofloxacin), tetracyclines (chlortetracycline, oxytetracycline), macrolides (tilmicosin, tylosin), and chloramphenicols (chloramphenicol, florfenicol). Example 1

[0025] 1. Sample pretreatment and spiking Ten 5.00g homogenized egg samples were accurately weighed and placed in 50mL centrifuge tubes. Five samples served as blank controls, while the other five were used for spiking. In the spiking experiment, a mixture of standard solutions of the ten target veterinary drugs was precisely added to each sample to ensure a final concentration of 50 µg / kg. The spiked samples were allowed to stand for 30 minutes to ensure sufficient contact between the drugs and the matrix.

[0026] Add 10.00 mL of pure acetonitrile (volume-to-mass ratio 2:1) to each 50 mL centrifuge tube, vortex for 2 minutes, and let stand at room temperature for 10 minutes.

[0027] Centrifuge at 4℃ and 10000 rpm for 8 minutes. Collect the supernatant and filter it through a 0.22 μm PTFE membrane to obtain the sample loading solution.

[0028] 2. Activation and Equilibrium of Solid Phase Extraction Column Waters Oasis HLB column (60 mg / 3 mL) was used.

[0029] Activation: Use 3.0 mL of pure methanol at a flow rate of 1 mL / min.

[0030] Equilibration: Use 3.0 mL of ultrapure water at a flow rate of 1 mL / min.

[0031] 3. Sample loading All the solution to be loaded was added to the solid-phase extraction column at a flow rate of 1 mL / min. After loading was complete, the column was dried under vacuum for 2 minutes.

[0032] 4. Multi-stage washing First washing: Use 2 mL of a 5% methanol aqueous solution with pH=4.0 at a flow rate of 0.8 mL / min, and then dry for 1 minute.

[0033] Second washing: Use 2 mL of a 20% acetonitrile aqueous solution with pH=7.0 at a flow rate of 0.8 mL / min, and then dry for 1 minute.

[0034] 5. Core synergistic elution S5-1: Use 1.0 mL of 0.1% formic acid aqueous solution containing 60% acetonitrile at pH=3.0, at a flow rate of 0.5 mL / min, to collect the first elution fraction.

[0035] S5-2: Inject 1.0 mL of 80% acetone aqueous solution at a flow rate of 0.5 mL / min, let stand for 1.0 minute, and discard the outflow.

[0036] S5-3: Collect the second elution fraction using 1.0 mL of 10 mM sodium acetate buffer containing 50% acetonitrile at pH 7.0 at a flow rate of 0.5 mL / min.

[0037] S5-4: Inject 1.0 mL of 90% acetone aqueous solution at a flow rate of 0.5 mL / min, let stand for 1.0 minute, and discard the outflow.

[0038] S5-5: Collect the third elution fraction using 1.0 mL of a 5 mM ammonium bicarbonate / 0.5% ammonia solution containing 90% acetonitrile at pH 9.0 at a flow rate of 0.5 mL / min.

[0039] Although the first competitive acetone pulse (S5-2) was performed after the first elution (S5-1), most of the free phospholipids in the egg sample had been removed after multiple washings (S4); and under acidic conditions (pH 2.5-4.0), the retention of phospholipids on the HLB adsorbent was weak and they flowed out with the washing solution. Therefore, the residual level of phospholipids in the first elution fraction was significantly reduced, ensuring a low matrix effect for sulfonamides and quinolones.

[0040] 6. Follow-up Analysis The collected first, second, and third elution fractions were concentrated to near dryness by nitrogen blowing at 40°C.

[0041] Before reconstitution, 5 μL of pure formic acid (final concentration 0.3%) was added to the third elution fraction to neutralize the alkaline environment and prevent chloramphenicol compounds from undergoing ring-opening degradation in subsequent treatments.

[0042] All components were reconstituted with 1.0 mL of 30% methanol aqueous solution, filtered through a 0.22 μm PTFE membrane, and then analyzed by LC-MS / MS.

[0043] Phospholipid residue fractional detection: During LC-MS / MS analysis, in addition to the target veterinary drug, the response intensity of the characteristic fragment ion of phosphatidylcholine (m / z 184.0732) in each elution fraction was monitored simultaneously, and the phospholipid removal efficiency of the first, second and third elution fractions was calculated respectively.

[0044] Liquid chromatography conditions: Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm); column temperature 40°C; injection volume 5 µL; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution program: 0–1 min, 10% B; 1–3 min, 10% B linearly increased to 40% B; 3–6 min, 40% B linearly increased to 80% B; 6–7 min, 80% B maintained; 7–7.1 min, 80% B linearly recovered to 10% B; 7.1–9 min, 10% B equilibrated.

[0045] Mass spectrometry conditions: Electrospray ionization (ESI), positive ion mode (for most veterinary drugs), negative ion mode (for chloramphenicol); capillary voltage 3.0 kV; cone voltage 30 V; ion source temperature 150°C; desolvation gas flow rate 1000 L / hr, desolvation gas temperature 500°C. Quantitative analysis was performed using multiple reaction monitoring (MRM) mode. Two characteristic ion pairs were selected for each target analyte: one for quantification and one for qualitative confirmation. Example 2

[0046] The parameters, conditions, and processing in this embodiment are basically the same as those in Embodiment 1, except for the following parameters.

[0047] 1. Sample pretreatment The volume-to-mass ratio of acetonitrile to egg sample was 3:1.

[0048] Centrifuge at 4℃ and 12000 rpm for 5 minutes.

[0049] 2. Activation and Equilibrium of Solid Phase Extraction Column Activation / equilibration: Use 5.0 mL of pure methanol and 5.0 mL of ultrapure water at a flow rate of 2 mL / min.

[0050] 3. Sample loading flow rate: 2 mL / min.

[0051] 4. Washing: First section: 2 mL of a 10% methanol aqueous solution with pH=5.0.

[0052] Second part: 2 mL of an aqueous solution containing 30% acetonitrile with pH=8.0.

[0053] 5. Core synergistic elution S5-1: 2.0 mL of eluent containing 80% acetonitrile at pH 4.0, at a flow rate of 1 mL / min.

[0054] S5-2: 2.0 mL 90% acetone, flow rate 1 mL / min, let stand for 2.0 minutes.

[0055] S5-3: 2.0 mL of eluent containing 60% acetonitrile at pH 8.0, at a flow rate of 1 mL / min.

[0056] S5-4: 2.0 mL 95% acetone, flow rate 1 mL / min, let stand for 2.0 minutes.

[0057] S5-5: 2.0 mL of 95% acetonitrile eluent at pH 10.0, at a flow rate of 1 mL / min. Example 3

[0058] The parameters, conditions, and processing in this embodiment are basically the same as those in Embodiment 1, except for the following parameters.

[0059] 1. Sample pretreatment The volume-to-mass ratio of acetonitrile to egg sample was 1:1.

[0060] Centrifuge at 4℃ and 8000 rpm for 10 minutes.

[0061] 2. Activation and Equilibrium of Solid Phase Extraction Column Activation / equilibration: Use 2.0 mL of pure acetonitrile and 2.0 mL of ultrapure water at a flow rate of 0.5 mL / min.

[0062] 3. Sample loading Flow rate: 0.5 mL / min.

[0063] 4. Multi-stage washing: First section: 2 mL of a 1% methanol aqueous solution with pH=3.0.

[0064] Second part: 2 mL of an aqueous solution containing 10% acetonitrile with pH=6.0.

[0065] 5. Core synergistic elution S5-1: 0.5 mL of eluent containing 50% acetonitrile at pH 2.5, at a flow rate of 0.2 mL / min.

[0066] S5-2: 0.5 mL 60% acetone, flow rate 0.2 mL / min, let stand for 0.5 minutes.

[0067] S5-3: 0.5 mL of eluent containing 40% acetonitrile at pH 6.5, at a flow rate of 0.2 mL / min.

[0068] S5-4: 0.5 mL 70% acetone, flow rate 0.2 mL / min, let stand for 0.5 minutes.

[0069] S5-5: 0.5 mL of eluent containing 80% acetonitrile at pH 8.5, at a flow rate of 0.2 mL / min.

[0070] Comparative Example 1: Traditional single-wash elution The sample pretreatment, activation / equilibration, and loading steps of Comparative Example 1 were exactly the same as those of Example 1.

[0071] Single wash: Only one wash solution is used, consisting of a 5% methanol aqueous solution (pH 7.0), with a volume of 5.0 mL and a flow rate of 1.0 mL / min.

[0072] Single gradient elution: A single eluent was used, consisting of an 80% acetonitrile aqueous solution (containing 0.1% formic acid), with a volume of 5.0 mL and a flow rate of 0.8 mL / min.

[0073] Comparative Example 2: pH gradient elution without acetone pulses All steps in this comparative example are exactly the same as in Example 1, except that the two acetone pulse injections at S5-2 and S5-4 are omitted.

[0074] Data Analysis and Comparison The recoveries of each veterinary drug were calculated by comparing the analytical results of spiked samples and blank samples from Examples 1-3 and Comparative Examples 1-2. Simultaneously, the matrix effect (ME) was evaluated by comparing the responses of spiked samples in the blank matrix and pure solvent. Phospholipid removal efficiency was assessed by LC-MS / MS analysis of the peak area changes of characteristic phospholipid ions (m / z 184.0732, characteristic fragment ion of phosphatidylcholine) in each eluent fraction. Table 1 summarizes the recovery rate data of 10 veterinary drugs from Examples 1-3 and Comparative Examples 1-2; Table 2 summarizes the matrix effect data of 10 veterinary drugs from Examples 1-3 and Comparative Examples 1-2; and Table 3 summarizes the phospholipid removal efficiency (%) data of each eluent fraction from Examples 1-3 and Comparative Examples 1-2.

[0075] Table 1 Comparison of recovery rates (%) of 10 veterinary drugs

[0076] As shown in Table 1, the present invention (Examples 1-3) achieves high and balanced recovery rates for multiple categories of veterinary drugs, effectively solving the problems of uneven recovery rates and low recovery rates for some drugs in traditional methods. Moreover, the recovery rates for all target veterinary drugs are significantly higher than those of Comparative Example 1 and Comparative Example 2.

[0077] The recoveries of the 10 representative veterinary drugs in Examples 1-3 ranged from 78.8% to 93.0%, meeting the recovery rate requirements for multi-residue analysis. This indicates that the three-stage pH gradient elution creates an optimal elution environment for drugs with different pKa and polarities, ensuring that acidic, neutral, and basic drugs can be eluted efficiently.

[0078] The recovery rates in Comparative Example 1 showed a significant divergence (55.3%-80.3%). Specifically, the recovery rates of drugs such as oxytetracycline and chlortetracycline were below 60%, failing to meet the detection requirements. This demonstrates that a single elution condition cannot accommodate multiple drugs with varying properties. Using a strong elution buffer to elute strongly retaining drugs can lead to the loss of weakly retaining drugs in earlier steps; conversely, a weak elution buffer will prevent the elution of strongly retaining drugs.

[0079] Although the recovery rate of Comparative Example 2 (74.1%-88.1%) was better than that of Comparative Example 1, it was significantly lower than that of Examples 1-3. This demonstrates that competitive acetone pulses are not only used for phospholipid removal, but their role in purifying the column environment and releasing phospholipid-occupied adsorption sites is also crucial for achieving high recovery rates for all target analytes. The absence of this step can lead to incomplete drug recovery.

[0080] Table 2 Comparison of matrix effects (%) of 10 veterinary drugs

[0081] As shown in Table 2, the method of the present invention (Examples 1-3) reduces the matrix inhibition effect to a low level by deeply removing phospholipids, which significantly improves the accuracy of quantification and detection sensitivity.

[0082] The method of this invention (Examples 1-3) successfully controlled the matrix effect within a weak range of -4.5% to -18.5%. This data indicates that the LC-MS / MS response of samples treated by the method of this invention is relatively close to that in pure solvents, and most of the ion inhibition caused by interfering substances such as phospholipids has been essentially eliminated. This lays a solid foundation for the accurate quantification of trace (µg / kg level) veterinary drugs.

[0083] Comparative Example 1 exhibited a strong ion suppression effect (-20.1% to -41.2%). This means that the target analyte signal is severely suppressed, which can lead to a significant decrease in detection sensitivity, significantly lower quantitative results, and even false negatives.

[0084] Although the matrix effect (-13.7% to -35.8%) in Comparative Example 2 was improved compared to Comparative Example 1, it was still quite strong. This directly proves that phospholipids are the main culprit for the matrix effect, and that pH gradient elution alone cannot effectively solve the problem of phospholipid co-elution. The two competitive acetone pulses in this invention are a key technical means to achieve deep phospholipid removal and thus overcome the matrix effect.

[0085] Table 3 Comparison of phospholipid removal efficiency (%) of each elution component

[0086] As shown in Table 3, the method of the present invention (Examples 1-3) achieves the removal of most of the phospholipids in the egg matrix, which is the fundamental reason for achieving high recovery rate and low matrix effect.

[0087] The phospholipid removal efficiency of the method of the present invention (Examples 1-3) is consistently above 96%. This effect fundamentally cuts off the source of phospholipid interference and is the physical basis for achieving the aforementioned excellent performance (high recovery rate, low matrix effect).

[0088] The phospholipid removal efficiencies of Comparative Examples 1 and 2 were both unsatisfactory (Comparative Example 1: 65.5%; Comparative Example 2: 73.8%-78.5%), with a large amount of phospholipids remaining in the final samples. These residual phospholipids not only clog the ion source and reduce instrument stability in LC-MS / MS analysis, but more importantly, they cause severe ion inhibition, resulting in a significant decrease in the detection sensitivity of trace target analytes and potentially leading to false negative results. The competitive acetone pulse injection technology of this invention, through the ingenious insertion of a competitive binding between acetone and the adsorbent during the elution process, successfully elutes and discards stubborn phospholipids from the solid-phase extraction column, solving this long-standing problem of detecting veterinary drug residues in animal-derived samples (especially egg samples).

[0089] In summary, the solid-phase extraction process for detecting veterinary drug residues in animal-derived samples described in this invention achieves significant improvements in recovery rate, matrix effect control, and phospholipid removal efficiency through the organic combination and synergistic effect of three-stage pH gradient elution and competitive acetone pulse injection. This provides an efficient, reliable, and reproducible pretreatment solution for the accurate and highly sensitive detection of complex and multi-category veterinary drug residues in animal-derived samples (especially egg samples).

[0090] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A solid-phase extraction process for detecting veterinary drug residues in animal-derived samples, characterized in that: Includes the following steps: S1. Sample pretreatment: The homogenized animal-derived sample is mixed with acetonitrile, and protein precipitation, centrifugation and filtration are performed to obtain the sample loading solution. S2. Solid-phase extraction column activation and equilibration: A hydrophilic-lipophilic balanced polymer adsorbent solid-phase extraction column is used, which is activated by organic solvent and equilibrated by ultrapure water in sequence. S3. Sample loading: The sample solution is loaded onto the solid-phase extraction column after activation equilibrium. After the sample solution has completely flowed through the solid-phase extraction column, it is dried. S4. Multi-stage washing: The solid phase extraction column is washed in stages using washing solutions composed of at least two different pH values ​​and organic solvents; S5, Core Synergistic Elution: The following sub-steps are performed sequentially by combining three-stage pH gradient elution with two competitive acetone pulse injections: S5-1, First stage pH gradient elution: Elute using an eluent with a pH of 2.5-4.0 and containing 50%-80% organic solvent, and collect the first eluted fraction; S5-2, First competitive acetone pulse injection: Inject an acetone aqueous solution with a concentration of 60%-90% into the solid phase extraction column, and discard the effluent after standing. S5-3, Second stage pH gradient elution: Elute using an eluent with a pH of 6.5-8.0 and containing 40%-60% organic solvent, and collect the second eluted fraction; S5-4, Second competitive acetone pulse injection: Inject an acetone aqueous solution with a concentration of 70%-95% into the solid phase extraction column, and discard the effluent after standing; S5-5, Third-stage pH gradient elution: Elution is performed using an eluent with a pH of 8.5-10.0 and containing 80%-95% organic solvent, and the third eluted fraction is collected.

2. The method according to claim 1, characterized in that: In step S1, the volume-to-mass ratio of the acetonitrile to the animal-derived sample is 1:1 to 3:

1.

3. The method according to claim 1, characterized in that: In step S1, the centrifugation conditions are 4°C and 8000-12000 rpm for 5-10 minutes.

4. The method according to claim 1, characterized in that: In step S1, the filtration is performed using a 0.22 μm polytetrafluoroethylene filter membrane.

5. The method according to claim 1, characterized in that: In step S2, the organic solvent used for activation is methanol or acetonitrile; wherein the amount of the organic solvent added is 2.0-5.0 mL, and its flow rate is 0.5-2 mL / min.

6. The method according to claim 1, characterized in that: In step S3, the flow rate of the solution to be loaded is 0.5-2 mL / min.

7. The method according to claim 1, characterized in that: In step S4, the multi-stage washing includes: First washing: Use a washing solution with a pH of 3.0-5.0 and containing 1%-10% methanol for washing; Second washing: Use a washing solution with a pH of 6.0-8.0 and containing 10%-30% acetonitrile for washing.

8. The method according to claim 1, characterized in that: In step S5, the volume of the first and second competitive acetone pulse injections is independently 0.5-2 mL, the injection flow rate is 0.2-1 mL / min, and the residence time in the solid phase extraction column is 0.5-2 min. The organic solvent in the first, second, and third pH gradient eluents is acetonitrile; wherein the volume of the eluent injected is independently 0.5-2 mL, and the injection flow rate is 0.2-1 mL / min.

9. The method according to claim 1, characterized in that: The animal-derived sample was an egg sample.

10. The method according to any one of claims 1-9, characterized in that: The method also includes subsequent analysis steps: the first, second and third elution fractions are concentrated and reconstituted respectively, and then qualitative and quantitative analysis is performed by liquid chromatography-tandem mass spectrometry; wherein, the third elution fraction is acidified by adding an acidic solution before concentration.

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