Method for rapidly determining 17 novel veterinary drug compounds in animal-derived food through combination of solid-phase extraction and HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method

Abstract

The invention relates to the technical field of food safety detection, discloses a method for rapidly determining 17 novel veterinary drug compounds in animal-derived food by combining solid-phase extraction with HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), and relates to an HPLC-MS / MS method for determining 17 novel veterinary drug residues such as yohimbine, medetomidate and tripinramine in chicken, beef, fish and shrimp. Trade barriers and economic losses caused by drug residues can be avoided; comprising the following steps: 1) preparing standard stock solutions with different concentrations by using standard substances of 17 novel veterinary drug compounds, and mixing standard working stock solutions; a series of working standard solutions are prepared by using the standard stock solution and are used for HPLC-MS / MS detection; 2) adding the mixed standard working stock solution into extracted and purified blank sample residues to prepare a series of matrix matching mixed standard solutions with different concentrations, filtering the mixed standard solutions, and loading the mixed standard solutions on a machine for detection, and 3) homogenizing the samples of cattle, chicken, fish and prawns, carrying out subsequent treatment, and carrying out HPLC-MS / MS online analysis on the obtained samples.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a method for rapidly determining 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS. Background Art

[0002] Veterinary drugs are commonly used to treat and control diseases and are used as growth promoters and feed enhancers in livestock production (Adou et al. 2001, Bacanli et al. 2019). Recently, cases of improper and excessive use of veterinary drugs have been documented. It is estimated that about 80% of food animals are treated with veterinary drugs for part or most of their lives (Lee et al. 2001). Long-term consumption of these products may have a range of adverse effects on human health, including allergic reactions, carcinogenesis, organ dysfunction, antimicrobial resistance and other harmful consequences (Hosain et al. 2021, Ferdous et al. 2019). There are many types of veterinary drugs in animal-derived foods, and the relevant standards and limit requirements are updated rapidly. Yohimbine is an α2-adrenergic receptor antagonist used in the veterinary field to reverse the effects of α2-receptor agonists, xylazine and detomidine. (DiMaio Knych et al. 2011). However, current detection methods are mainly targeted at botanicals, barks, and dietary supplements (Sun et al. 2012, Lucas et al. 2015, Quiroz-Delfi et al. 2024), and there are few studies on the detection of this substance in animal-derived food matrices. The maximum residue limit of yohimbine in venison set by the Korean government is 0.01 mg / kg. In addition, there are not enough public reports involving the use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to detect medetomidine residues in animal-derived foods. However, the Korean government has set the maximum residue level (MRL) in pork at 0.01 mg / kg (Korean Ministry of Food and Drug Safety 2015). In a study conducted by Kim et al. (2021), Quechers and HPLC-MS / MS were combined to detect the residues of several drugs such as acetanilide, phenacetin, antipyrine, diethylcarbamazine, tripelennamine, and loperamide in fish and shrimp samples. This study did not involve the analysis of livestock matrices. However, it should be noted that the regulatory frameworks of South Korea and the United States have relatively strict requirements for these substances in animal muscle (Korean Ministry of Food and Drug Safety 2015, US Food and Drug Administration 2023). However, these substances are not yet covered by the Chinese standards GB 31650 and GB 31650.1. The pretreatment purification methods for veterinary drug residues are mostly based on solid phase extraction (SPE), dispersive solid phase extraction (DSPE), magnetic solid phase extraction (M-SPE), dispersive liquid-liquid microextraction (DLLME) and liquid-liquid extraction (LLE) techniques (Drabińska et al. 2023).Currently, solid phase extraction (SPE) cartridges are widely used, but during the pretreatment process, a series of operations such as cartridge activation, loading, washing and elution are time-consuming and require a large amount of reagents. Summary of the invention

[0003] The purpose of the present invention is to provide a method for rapidly determining 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, which is an HPLC-MS / MS method for determining 17 new veterinary drug residues such as yohimbine, medetomidine, and tripelennamine in chicken, beef, fish, and shrimp, and can avoid trade barriers and economic losses caused by drug residue problems. The method complies with the latest food safety regulations and limit requirements in South Korea, Canada, and the United States.

[0004] The present invention is achieved through the following technical scheme: a method for rapidly determining 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, comprising the following steps:

[0005] 1) Standard stock solutions of 17 new veterinary drug compounds were prepared with different concentrations and mixed standard working stock solutions; and a series of working standard solutions were prepared using the standard stock solutions for HPLC-MS / MS detection;

[0006] 2) Add the mixed standard working stock solution to the blank sample residue after extraction and purification to prepare a series of matrix-matched mixed standard solutions of different concentrations, filter them and put them on the machine for testing;

[0007] 3) The samples of beef (muscle), chicken (muscle), fish (fish skin and muscle), and shrimp (muscle) were homogenized and subsequently processed, and the obtained samples were subjected to HPLC-MS / MS online analysis.

[0008] In order to better implement the method of the present invention for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, the following configuration is particularly adopted: Step 1) includes the following specific steps:

[0009] 1.1) Accurately weigh 0.01 g of butaquinate standard in a 10 mL volumetric flask, add methanol to dissolve and dilute to scale; prepare a standard stock solution with a mass concentration of 1000 μg / mL, and store it at -20°C away from light;

[0010] 1.2) Use a pipette to accurately transfer 1 mL of each of the remaining 16 standards, add methanol or acetonitrile to make the final volume of each standard reach 10 mL; prepare standard stock solutions of the remaining standards with a mass concentration of 10 μg / mL or 100 μg / mL, and store them in a dark environment at -20°C for future use;

[0011] 1.3) Accurately measure an appropriate amount of standard stock solution and dilute it with methanol to prepare a mixed standard working stock solution with a mass concentration of 1 μg / mL; then, prepare a series of working standard solutions with concentrations of 2, 4, 10, 20, 50, 200 and 400 ng / mL for HPLC-MS / MS detection.

[0012] In order to better implement the method of the present invention for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, the following configuration is particularly adopted: Step 2) is specifically as follows:

[0013] The mixed standard working stock solution was quantitatively added to 7 portions of blank sample residues after extraction and purification, and dried under nitrogen environment at 45°C; 1 mL of methanol was vortexed to dissolve, and a matrix-matched series of mixed standard solutions were prepared, which were filtered through a 0.22 μm filter membrane for testing; a standard curve was drawn with the measured characteristic ion peak area as the ordinate and the corresponding standard solution concentration as the abscissa.

[0014] In order to better realize the method of the present invention for rapidly determining 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, the following setting is particularly adopted: the concentrations of the matrix-matched series of mixed standard solutions are 2, 4, 10, 20, 50, 200 and 400 ng / mL, respectively.

[0015] In order to further better implement the method of the present invention for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, the following configuration is particularly adopted: the step 3) specifically includes the following steps:

[0016] 3.1) The samples of beef (muscle), chicken (muscle), fish (skin and muscle), and shrimp (muscle) were homogenized in a homogenizer and stored at -20°C;

[0017] 3.2) Accurately weigh 2 g of the homogenized sample in a 50 mL plastic centrifuge tube, add 8 mL of acetonitrile: water (85:15) and 1 g of Ma2SO4; shake the sample for 5 min, mix thoroughly, and then extract it in an ultrasonic water bath for 15 min; then freeze it and centrifuge it at 10,000 rpm and 4°C for 5 min;

[0018] 3.3) After step 3.2), the supernatant was loaded into an HLB column, and then all the eluates were collected; subsequently, 2 mL of acetonitrile was added for washing; the two eluates were combined and evaporated to dryness under nitrogen at 45°C; 1 mL of methanol was accurately added, the residue was vortexed to dissolve, and the residue was filtered through a 0.22 μm microporous membrane for HPLC-MS / MS online analysis.

[0019] In order to better implement the method of the present invention for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, the following setting is particularly adopted: in the step 3.2), the ratio of acetonitrile to water is 85:15.

[0020] In order to better implement the method of the present invention for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, the following configuration is particularly adopted: when performing HPLC-MS / MS online analysis,

[0021] The chromatographic conditions are:

[0022] The chromatographic column Acquity UPLC HSS T3 (2.1 mm*100 mm, 1.8 μm, Waters Corporation, USA) was used for separation; the mobile phase A was methanol, and the mobile phase B was 0.2% formic acid water; the elution conditions of the veterinary drug residue mixture were: 5% A at 0–2 min, 5-90% A at 2–10 min, 90–95% A at 10–10.1 min, 95% A at 10.1–12 min, 95–5% A at 12–12.5 min, 5% A at 12.5–15 min; the total duration of the experiment was 15 min. In addition, the flow rate was 0.25 mL / min, and the injection volume was 2.0 μL;

[0023] The mass spectrometry conditions were:

[0024] Electrospray positive / negative ion (ESI+ / -) mode was used; the electrospray capillary voltage was set to 3.10 kV (ESI+) and 2.50 kV (ESI-), and the ESI cone voltage was set to 67 V (+) and 20 V (-); under the condition of a dissolution temperature of 450 ° C, the dissolved gas flow rate of ESI+ and ESI- was 800 L / h and 600 L / h, respectively, and the collision gas flow rate was 0.15 mL / min. In addition, qualitative analysis of each compound was performed under the multiple reaction monitoring mode (MRM).

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] Unlike traditional solid phase extraction methods, the present invention adopts straight-through solid phase extraction technology. Oasis HLB cartridges are used for sample purification without activation / equilibrium and elution. The sample solution is directly loaded onto the solid phase extraction cartridge, which is a more direct process compared to traditional solid phase extraction cartridges. Oasis HLB cartridge is a commonly used SPE cartridge composed of a macroporous copolymer of two monomers: lipophilic divinylbenzene and hydrophilic N-vinylpyrrolidone, which are polymerized in a specific ratio. It is suitable for extracting and purifying target compounds from complex samples. The cartridge is based on the HLB (hydrophobic lipophilic balance) principle, which can effectively adsorb and remove lipid and protein components in the sample, thereby promoting the purification and enrichment of the target compound.

[0027] The present invention uses a pass-through SPE pretreatment technology to develop an HPLC-MS / MS method for determining 17 new veterinary drug residues in animal-derived foods. The present invention optimizes influencing factors, including mass spectrometry parameters, mobile phase, extraction solvent, SPE column type and extraction solvent dosage. Evaluation indicators include matrix effect, linearity, recovery rate and precision. The method is simple, efficient and economical, and has been successfully applied to actual sample testing in local markets. It can provide valuable technical support for strengthening cross-border food safety and eliminating trade barriers.

[0028] The present invention establishes a method for rapid determination of 17 new veterinary drug residues in animal-derived foods by HPLC-MS / MS. The optional straight-through SPE Oasis HLB kit facilitates efficient sample cleanup without the need for activation / equilibrium and elution. The results show that the established HPLC-MS / MS method has good performance. The recovery of the test was between 65.1-114.4%, and the relative standard deviation (RSD) was less than 12.3%. The limit of quantification (LOQ) of the target compound was in the range of 0.3-2.0 μg / kg (S / N ≥ 10), which meets the limit requirements of the relevant matrix in the food safety standards of South Korea, the United States and Canada in recent years. At present, there are few literatures on the detection of 17 target veterinary drug residues in animal-derived foods. Compared with existing reports, the method proposed in the present invention is relatively simple, highly sensitive, and applicable to a variety of matrices. It has been successfully used to detect a variety of animal-derived foods in the local market, thus providing valuable technical assistance for cross-border food safety testing. Pentylenetetrazol-positive samples were found in two batches of fish and one batch of chicken. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 17 target veterinary drug residues (20 ng / ml) were effectively separated on the chromatogram.

[0030] Figure 2 This is a graph showing the effect of extraction solvent on the recovery rates of 17 target veterinary drugs (taking shrimp as an example).

[0031] Figure 3 To study the effect of extraction solvents on the recovery rates of 17 target veterinary drugs (taking chicken as an example).

[0032] Figure 4 This is a graph showing the effect of extraction solvent on the recovery rates of 17 target veterinary drugs (taking cattle as an example).

[0033] Figure 5 This is a graph showing the effect of extraction solvent on the recovery rates of 17 target veterinary drugs (taking fish as an example).

[0034] Figure 6 Effect of optimized solid phase extraction column on the recovery of 17 target veterinary drugs.

[0035] Figure 7 Schematic diagram of the effect of different extraction solvent dosages on the recovery of target veterinary drugs in fish meat.

[0036] Figure 8 Figure 2 shows the effect of two salts in fish matrix on the recovery of target veterinary drugs.

[0037] Fig. 9 This is a graph showing the effects of two salts in shrimp matrix on the recovery of target veterinary drugs.

[0038] Fig.10 Effect of two salts on the recovery of target veterinary drugs in bovine matrix.

[0039] Fig.11 Effect of two salts on the recovery of target veterinary drugs in chicken matrix. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0041] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0042] Glossary:

[0043] In the drawings given in the present invention, Acetanilide is acetanilide, Pentetrazol is pentylenetetrazol, Methylephedrine hydrochloride is methylephedrine hydrochloride, Phenacetin is phenacetin, Antipyrine (Phenazone) is antipyrine, Guaifenesin is guaifenesin, Diethylcarbamazine is diethylcarbamazine, Acridinium yellow (8048-52-0) is a mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-acridinediamine, Metomidate is metomidate, Acrinol is rivanol, Tripelennamine hydrochloride is tripelennamine, Ormetoprim is ormetoprim, Sulfaethoxypyridazine is sulfaethoxypyridazine, Yohimbine hydrochloride is yohimbine, Buquinolate is butyquinolate, Loperamide hydrochloride is loperamide, Bithionol is thiobisdichlorophene, Acetonitrile is acetonitrile, 0.1% formic acid acetonitrile is 0.1% formic acid acetonitrile, and 0.2% ammoniaacetonitrile is 0.2% ammonia acetonitrile.

[0044] In order to achieve better technical effects, the present invention involves the following instruments, materials and reagents:

[0045] HPLC system (HPLC 1-class-XEVOTQ-XS, Waters Corporation, USA); chromatographic column Acquity UPLC HSS T3 (2.1 mm*100 mm, 1.8 μm, Waters Corporation, USA).

[0046] All HPLC grade methanol and acetonitrile were provided by CNW Technologies GmbH (Düsseldorf, Germany). Ammonium acetate, formic acid (FA), and ammonium hydroxide (HPLC grade) were provided by Chengdu Kelong Chemical Co., Ltd. (Sichuan, China). Analytical grade sodium chloride (NaCl), anhydrous sodium sulfate (Na2SO4), and C18 adsorbent (40-63 μm, C18) were purchased from Anpel Laboratory Technologies (Shanghai, China). The water purification system was from the Mili-Q Integral 3 ultrapure water equipment produced by Millipore (Boston, USA). Cleanert S C18 (60 mg / 3 ml 50 / pkg) was provided by Agela Technology (Shanghai, China). Oasis HLB cartridges (3 cc / 60 mg and 6 cc / 500 mg) and Oasis PRiME HLB cartridges (3 cc / 60 mg) were purchased from Waters (Milford, USA). Butylquinac (10 mg, 98.3%, CAS 5486-03-3) was purchased from Beijing Manhai Biotechnology Co., Ltd., China. All other standard materials were liquid standard materials with a purity of ≥94.6% from AltaScientific Co. (Tianjin, China). Table 1 lists more detailed information on the target veterinary drug residues, including Chemical Abstracts Service (CAS) number, molecular weight, solvent, and chemical structure. It is worth noting that, as shown in the purchased standards, acriflavine is a mixture of a known parent compound (3,6-diamino-10-methylacridinium chloride) and a demethylated derivative, acriflavine (Turnipseed et al. 2019), which showed different peaks and retention times on the chromatographic column. Therefore, in subsequent analysis, these substances were treated as two different substances.

[0047] Table 1 CAS number, molecular weight, solvent, chemical structure and pharmacological use of 17 veterinary residues

[0048]

[0049]

[0050]

[0051] * Commercially available acridine yellow is a mixture of 3,6-diamino-10-methylacridinium chloride and 3,6-Acridinediamine (Proflavine).

[0052] Embodiment 1:

[0053] A method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS comprises the following steps:

[0054] 1) Standard stock solutions and mixed standard working stock solutions of different concentrations were prepared using standard substances of 17 new veterinary drug compounds; and a series of working standard solutions were prepared using the standard stock solutions for HPLC-MS / MS detection; among them, the 17 new veterinary drug compounds were: acetanilide, pentylenetetrazol, methylephedrine hydrochloride, phenacetin, antipyrine, guaifenesin, ethodicarbamazine, acriflavine (a mixture of chloro-3,6-diamino-10-methylacridine and acridinium-3,6-diamine), medetomidine, rivanol, tripeliramine, ormeprim, sulfathionethoxyfen, yohimbine, butaquinate, loperamide and dichlorophenthiophene.

[0055] 2) Add the mixed standard working stock solution to the blank sample residue after extraction and purification to prepare a series of matrix-matched mixed standard solutions of different concentrations, filter them and put them on the machine for testing;

[0056] 3) The samples of beef (muscle), chicken (muscle), fish (fish skin and muscle), and shrimp (muscle) were homogenized and subsequently processed, and the obtained samples were subjected to HPLC-MS / MS online analysis.

[0057] Embodiment 2:

[0058] This embodiment is further optimized on the basis of the above embodiment, and the same points as the above technical solution are not repeated here. In order to better realize the method of rapid determination of 17 new veterinary drug compounds in animal-derived food by solid phase extraction combined with HPLC-MS / MS method described in the present invention, the following setting is particularly adopted: the step 1) includes the following specific steps:

[0059] 1.1) Accurately weigh 0.01 g of butaquinate standard in a 10 mL volumetric flask, add methanol to dissolve and dilute to scale; prepare a standard stock solution with a mass concentration of 1000 μg / mL, and store it at -20°C away from light;

[0060] 1.2) Use a pipette to accurately transfer 1 mL of each of the remaining 16 standards, add methanol or acetonitrile to make the final volume of each standard reach 10 mL; prepare standard stock solutions of the remaining standards with a mass concentration of 10 μg / mL or 100 μg / mL, and store them in a dark environment at -20°C for future use;

[0061] 1.3) Accurately measure an appropriate amount of standard stock solution and dilute it with methanol to prepare a mixed standard working stock solution with a mass concentration of 1 μg / mL; then, prepare a series of working standard solutions with concentrations of 2, 4, 10, 20, 50, 200 and 400 ng / mL for HPLC-MS / MS detection.

[0062] Embodiment 3:

[0063] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS method described in the present invention, the following setting is particularly adopted: the step 2) is specifically:

[0064] The mixed standard working stock solution was quantitatively added to 7 portions of blank sample residues after extraction and purification, and dried under nitrogen environment at 45°C; 1 mL of methanol was vortexed to dissolve, and a matrix-matched series of mixed standard solutions were prepared, which were filtered through a 0.22 μm filter membrane for testing; a standard curve was drawn with the measured characteristic ion peak area as the ordinate and the corresponding standard solution concentration as the abscissa.

[0065] Embodiment 4:

[0066] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS as described in the present invention, the following setting is particularly adopted: the concentrations of the matrix-matched series of mixed standard solutions are 2, 4, 10, 20, 50, 200 and 400 ng / mL, respectively.

[0067] Embodiment 5:

[0068] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS method described in the present invention, the following setting is particularly adopted: the step 3) specifically includes the following steps:

[0069] 3.1) The samples of beef (muscle), chicken (muscle), fish (skin and muscle), and shrimp (muscle) were homogenized in a homogenizer and stored at -20°C;

[0070] 3.2) Accurately weigh 2 g of the homogenized sample in a 50 mL plastic centrifuge tube, add 8 mL of acetonitrile: water (85:15) and 1 g of Ma2SO4; shake the sample for 5 min, mix thoroughly, and then extract it in an ultrasonic water bath for 15 min; then freeze it and centrifuge it at 10,000 rpm and 4°C for 5 min;

[0071] 3.3) After step 3.2), the supernatant was loaded into an HLB column, and then all the eluates were collected; subsequently, 2 mL of acetonitrile was added for washing; the two eluates were combined and evaporated to dryness under nitrogen at 45°C; 1 mL of methanol was accurately added, the residue was vortexed to dissolve, and the residue was filtered through a 0.22 μm microporous membrane for HPLC-MS / MS online analysis.

[0072] In order to achieve better detection results, the present invention has made the following optimization treatments on sample pretreatment technology during the research and development stage:

[0073] 1. Optimization of extraction solvent:

[0074] The selection of appropriate extraction reagents (extraction solvents) is crucial to ensure the effective recovery of various veterinary drug residues. Acetonitrile is one of the most commonly used extraction solvents in animal-derived foods. At the same time, the acidity and alkalinity of the extraction environment will also affect the extraction efficiency of the target substance. In the research and development stage, the present invention selected 0.1% formic acid acetonitrile, 0.2% ammonia acetonitrile, acetonitrile and acetonitrile: water (85:15) as extraction solvents to compare the extraction effects. Figure 2 to Figure 5 As shown in the figure, under the extraction conditions of 0.1% formic acid in acetonitrile and 0.2% ammonia in acetonitrile, the recovery of target veterinary drug residues is generally high, among which the recovery of methylephedrine in shrimp exceeds 140%, and the recovery of other substances is mostly between 110% and 130%. Acetonitrile has good solubility and strong permeability, which can precipitate proteins and effectively avoid excessive extraction of lipids, so it has the advantage of being able to extract target substances better than other solvents in most matrices. However, proteins are easy to agglutinate in organic solvents. Adding an appropriate amount of purified water to the extraction reagent can reduce the proportion of acetonitrile, promote sample dispersion, and slow down uniform agglutination. In addition, acetonitrile has limited ability to extract polar analytes. In contrast, aqueous solutions have a stronger ability to extract polar compounds. In summary, 85%:15% acetonitrile-water solution was selected as the best extraction solvent, showing excellent extraction efficiency, which meets the standards of Oasis HLB cartridges because the cartridges require a high proportion of organic phase for purification.

[0075] 2. The solid phase extraction column was optimized:

[0076] The matrix of animal samples is complex, and the extract contains a large amount of protein and fat-soluble impurities, which have an adverse effect on the stability of mass spectrometry analysis. Injecting the entire batch of samples into the system will have an adverse effect on the ion source, so the extract must be purified. Figure 6 The purification results of four solid phase extraction columns (Cleanert SC18, Oasis@HLB 3cc, Oasis@HLB6cc, Oasis@PRiME HLB 3cc) were compared. The extraction efficiency of the C18 columns of the four matrices is very high, and the extraction efficiency of some substances even exceeds 120%. HLB 6cc has a larger tube diameter and more fillers, so the filtration efficiency is higher and the pretreatment time is shorter. However, the recovery rate of the target veterinary drug residues is relatively low, especially for thiobisdichlorophene in the four matrices, the recovery rate is less than 40%. The results of HLB 3cc and Prime HLB are comparable, and HLB 3cc is slightly better in removing impurities (including fat and protein) in the extracted samples. Therefore, the present invention chooses to use HLB 3cc columns.

[0077] 3. The dosage of the extractant was optimized accordingly:

[0078] Taking fish as an example, the effects of five different solvent extraction doses (4, 6, 8, 10 and 12 mL) on the recovery of target substances were evaluated. Figure 7 As shown. As the volume of the extraction solvent increases, the recovery rate of the target substance also increases. However, after exceeding 12mL, the recovery rates of most substances decreased, which may be because interfering factors such as other impurities in the matrix are introduced at the same time, thereby inhibiting the detection of the target substance. It is observed that when the volume of the extraction solvent is 8mL and 10mL, the extraction efficiency is relatively close. In order to shorten the loading time and improve the subsequent efficiency of nitrogen blowing, the extraction solvent volume finally selected by the present invention is 8mL.

[0079] 4. Optimization of extraction powder:

[0080] The presence of water will reduce the concentration rate. Salt is usually added during extraction to reduce the solubility of veterinary drugs in the aqueous phase by salting out, thereby increasing the veterinary drug content in the organic phase, promoting the separation of the aqueous phase and the organic phase, and reducing the amount of water in the organic phase. The effects of sodium sulfate (Na2SO4) and sodium chloride (NaCl) on the recovery rate of the target compound were evaluated ( Figure 8 to Figure 11 The results showed that although NaCl dehydration can improve the recovery of individual substances (such as methylephedrine and phenacetin in shrimp; yohimbine, butaquinate and loperamide in beef), the recovery of most target compounds under Na2SO4 conditions was better when evaluated across the entire matrix range. Therefore, 1g Na2SO4 was selected for the final extraction.

[0081] Embodiment 6:

[0082] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS method described in the present invention, the following setting is particularly adopted: in the step 3.2), acetonitrile: water is 85:15.

[0083] Embodiment 7:

[0084] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS method described in the present invention, the following setting is particularly adopted: when performing HPLC-MS / MS online analysis,

[0085] The chromatographic conditions are:

[0086] The chromatographic column Acquity UPLC HSS T3 (2.1 mm*100 mm, 1.8 μm, Waters Corporation, USA) was used for separation; the mobile phase A was methanol, and the mobile phase B was 0.2% formic acid water; the elution conditions of the veterinary drug residue mixture were: 5% A at 0–2 min, 5-90% A at 2–10 min, 90–95% A at 10–10.1 min, 95% A at 10.1–12 min, 95–5% A at 12–12.5 min, 5% A at 12.5–15 min; the total duration of the experiment was 15 min. In addition, the flow rate was 0.25 mL / min, and the injection volume was 2.0 μL;

[0087] The mass spectrometry conditions were:

[0088] Electrospray positive / negative ion (ESI+ / -) mode was used; the electrospray capillary voltage was set to 3.10 kV (ESI+) and 2.50 kV (ESI-), and the ESI cone voltage was set to 67 V (+) and 20 V (-); under the condition of a dissolution temperature of 450 ° C, the dissolved gas flow rate of ESI+ and ESI- was 800 L / h and 600 L / h, respectively, and the collision gas flow rate was 0.15 mL / min. In addition, qualitative analysis of each compound was performed under the multiple reaction monitoring mode (MRM).

[0089] Since a single standard solution (100 ng / mL) was selected for the experiment, the precursor ions of the analytes were determined in positive and negative ion modes. Based on the optimized detector voltage conditions, the two fragment ions with the highest response were selected as product ions. In the positive ion mode, most substances showed higher responses accompanied by the best peak shapes. Given that most target compounds contain nitrogen-containing groups in their molecular structures, they can easily form [M+H]+ adducts in aqueous solutions (Schug et al. 2003). Therefore, [M+H]+ was used as the parent ion for daughter ion scanning. In contrast, thiobisdichlorophenol showed higher responses in the negative mode. This may be attributed to the presence of specific functional groups in its structure, such as acidic functional groups (e.g., phenolic hydroxyl groups, etc.), which make it easier to dissociate protons and form negatively charged ions (Li et al. 2024). The optimal mass spectrometry parameters for each target substance are shown in Table 2.

[0090] Table 2 Retention time and optimal mass spectrometry operating parameters for target veterinary drugs

[0091]

[0092] *Quantitative ion

[0093] Since polar solvents (such as water, methanol, acetonitrile, etc.) are prone to electrochemical reactions in the nozzle, polar solvents are generally used in ESI-MS experiments. The present invention compared the peak shape behaviors of methanol and acetonitrile in the research and development stage. The elution capacity of methanol / water mobile phase is lower than that of acetonitrile / water mobile phase. Therefore, the retention time of the target veterinary drug residue is extended, which is conducive to the separation of chromatographic peaks. Finally, methanol is selected as the organic phase (mobile phase A).

[0094] During the research and development stage, 5mmol / L ammonium acetate, 5mmol / L ammonium acetate (0.1% formic acid), 0.1% formic acid and 0.2% formic acid were used as the mobile phase water phase (mobile phase B), and the separation effect and response differences of different water phases on each target were investigated. The addition of formic acid may enhance the peak shape and protonation ability, thereby promoting the formation of the [M+H]+ peak and enhancing the reaction. Compared with 0.1% formic acid, 0.2% formic acid has a higher response and good peak shape, so methanol-0.2% formic acid water was finally selected as the mobile phase. Under the optimal conditions, the target veterinary drug residues on the chromatogram were quickly and completely separated ( Figure 1 ).

[0095] Experimental verification:

[0096] 1. Matrix effect assessment:

[0097] The matrix effect (ME) refers to the fact that after the sample is extracted and purified, some co-extracted substances may affect the ionization efficiency of the target substance, resulting in enhanced or suppressed signal response of the target substance. During the electrospray process, the target compound may be affected by the ionization and transfer of impurities, co-extracted analytes or degradation products. In addition, the competition between the matrix and the analyte for the limited element charge on the droplet may interfere with the detection or recovery of the target substance. Therefore, the matrix effect of this method was preliminarily studied. The negative sample was extracted and cleaned up, and then the matrix-matched calibration solution was prepared. ME was evaluated by the formula: ME = (A matrix standard curve slope / B pure solvent standard curve slope - 1) × 100%. The matrix effect of different types of substances is different. ME < 0 indicates matrix suppression, and vice versa, it indicates matrix enhancement. The larger the absolute value, the stronger the matrix effect. When |ME| < 20%, it is a weak matrix effect, indicating that the matrix effect is not significant; when |ME| < 50%, it is a moderate matrix effect; when |ME| > 50%, it is a strong matrix effect, indicating that the matrix effect is significant (Hu et al. 2020). Typical matrices used in the present invention include poultry (chicken), livestock (beef) and aquatic (black fish, shrimp) matrices. These matrices were selected to evaluate the matrix effects of the test targets in different product categories. As shown in Table 3, the target substances showed different degrees of matrix enhancement or matrix inhibition in all matrices. Most of the target substances in the beef matrix showed matrix enhancement effects (ME>0), while other matrices mainly showed matrix inhibition effects. Except for a few substances (trimethoprim, sulfaethoxypyridazine and loperamide hydrochloride) that showed weak matrix effects in individual matrices, most other substances showed moderate (ME>20%) or strong matrix effects (ME>50%). Therefore, this study adopted the matrix matching standard curve external standard method to reduce the influence of matrix effects on the determination of actual samples and improve the quantitative accuracy of the method.

[0098] Table 3 Matrix effects of target analytes in four matrices

[0099]

[0100]

[0101] 2. Performance evaluation of the method:

[0102] By preparing a series of matrix standard working solutions (matrix matching series mixed standard solutions) with concentrations of 2, 4, 10, 20, 50, 200 and 400 ng / ml, matrix matching calibration curves for 17 target veterinary drug residues were constructed. The abscissa x represents the analyte concentration and the ordinate y represents the measured peak area. The results are shown in Table 4. The correlation coefficient (R 2) were all greater than 0.996, indicating a good linear relationship. When the added concentration was 0.1-0.6 μg / kg, the signal-to-noise ratios of the 17 target veterinary drug residue ion pairs were all greater than 3, meeting the detection limit requirements; when the added concentration was 0.3-2.0 μg / kg, the signal-to-noise ratios were all greater than 10, meeting the quantitative limit requirements. By comparison, it was found that the minimum detection limits of all analytes in this method were lower than the veterinary drug limit requirements in South Korea, Canada and the United States (Table 4). This shows that this method meets the routine detection requirements for veterinary drug residues in multi-matrix animal-derived foods.

[0103] Table 4. Validation parameters and maximum residue limits of target veterinary drugs in beef, chicken, shrimp and fish.

[0104]

[0105]

[0106]

[0107] In addition, the recovery and relative standard deviations (RSDs) were investigated to evaluate the accuracy and precision of the application of 17 veterinary drug residues. The method was validated according to the CODEX Guidelines (CAC / GL 71-2009) (Kim et al. 2021, Codex Alimentarius Commission 2013), with a recovery of 60-120% and a relative standard deviation of 20-30% or less as validation criteria for determining the recovery and RSD. 5, 10 and 20 μg / kg of analytes (50, 100, 200 μg / kg for ormeprim, sulfaethoxypyridazine and butaquinate) were added to the samples. Six parallel analyses were performed on three quality control samples on the same day and on three consecutive days. The results showed that satisfactory recoveries (65.1-114.4%) were achieved for the target animal residues, with RSD ≤ 12.3% as shown in Table 5 (n = 6). From the collected data it can be inferred that the established UHPLC-MS / MS method is precise and accurate.

[0108] Table 5 Average recoveries and RSDs of target veterinary drug residues at different addition levels, n = 6

[0109]

[0110]

[0111] 3. Practical application of this method:

[0112] The applicability of the optimized HPLC-MS / MS method was evaluated on authentic samples purchased from the local market. A total of 62 batches of samples were analyzed and quantified using matrix-matched analytical calibration curves. The results showed the presence of pentylenetetrazol in two batches of fish and one batch of chicken, with detection rates of 11.1% and 7.1%, respectively, and residue levels of 3.8-5.0μg / kg and 2.4μg / kg, respectively (Table 6). However, it did not exceed the limit requirements. The remaining substances were not detected.

[0113] Table 6 Actual test samples

[0114]

[0115] Technology comparison:

[0116] Compared with other technologies, the technology disclosed in the present invention:

[0117] Park et al. (2018) established an LC-MS / MS method for the simultaneous determination of mefenamic acid, butaquinate and diclofenac in pork, eggs and milk. In contrast, the present invention can simultaneously detect more types of veterinary drugs with good stability. Kim et al. (2021) used a method combining QuEChERS with HPLC-MS / MS to detect multiple veterinary drug residues in fish and shrimp. In contrast, the solid phase extraction-UPLC-MS / MS method established in the present invention has higher sensitivity, lower limit of quantification and wider applicable matrix range for the detection of substances such as phenacetin, antipyrine, ethiprozine, tripelennamine, yohimbine and loperamide. Potter et al. (2007) developed an LC-MS / MS method for the detection of sulfonamides and synergists in salmon muscle. Hong et al. (2022) developed an LC-MS / MS method for the determination of sulfonamides, trimethoprim, ormeprim and dacron in fish and shrimp. In contrast, the present invention has a higher detection sensitivity for ormeprim and a wider range of applicable matrices. Therefore, the straight-through SPE-UPLC-MS / MS method developed by the present invention has significant advantages in terms of substance type, quantity and matrix. The method is simple and easy to use and has high sensitivity.

[0118] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS, characterized in that: The steps include: 1) Standard stock solutions of 17 new veterinary drug compounds were prepared with different concentrations and mixed standard working stock solutions; and a series of working standard solutions were prepared using the standard stock solutions for HPLC-MS / MS detection; 2) Add the mixed standard working stock solution to the blank sample residue after extraction and purification to prepare a series of matrix-matched mixed standard solutions of different concentrations, filter them and put them on the machine for testing; 3) The samples of cattle, chicken, fish and shrimp were homogenized and subsequently processed, and the obtained samples were subjected to HPLC-MS / MS online analysis.

2. The method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS according to claim 1, characterized in that: The step 1) comprises the following specific steps: 1.1) Weigh 0.01 g of butaquinate standard in a 10 mL volumetric flask, add methanol to dissolve and dilute to scale; prepare a standard stock solution with a mass concentration of 1000 μg / mL and store it at -20°C away from light; 1.2) Use a pipette to transfer 1 mL of each of the remaining 16 standards, add methanol or acetonitrile to make the final volume of each standard reach 10 mL; prepare standard stock solutions of the remaining standards with a mass concentration of 10 μg / mL or 100 μg / mL, and store them in a dark environment at -20°C for future use; 1.3) Accurately measure an appropriate amount of standard stock solution and dilute it with methanol to prepare a mixed standard working stock solution with a mass concentration of 1 μg / mL; then, prepare a series of working standard solutions with concentrations of 2, 4, 10, 20, 50, 200 and 400 ng / mL for HPLC-MS / MS detection.

3. The method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS according to claim 1, characterized in that: The step 2) is specifically as follows: The mixed standard working stock solution was quantitatively added to 7 portions of blank sample residues after extraction and purification, and dried under nitrogen at 45°C. 1 mL of methanol was vortexed to dissolve and a matrix-matched series of mixed standard solutions was prepared. The solutions were filtered through a 0.22 μm filter membrane and tested on the machine.

4. The method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS according to claim 3, characterized in that: The concentrations of the matrix-matched series of mixed standard solutions are 2, 4, 10, 20, 50, 200 and 400 ng / mL, respectively.

5. The method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS according to claim 4, characterized in that: The step 3) specifically includes the following steps: 3.1) The samples of cattle, chicken, fish and shrimp were homogenized in a homogenizer and stored at -20°C; 3.2) Accurately weigh 2 g of the homogenized sample in a 50 mL plastic centrifuge tube, add 8 mL of acetonitrile: water and 1 g of Ma2SO4; shake the sample for 5 min, mix thoroughly, and then extract it in an ultrasonic water bath for 15 min; then freeze it and centrifuge it at 10,000 rpm and 4°C for 5 min; 3.3) After step 3.2), the supernatant was loaded into an HLB column, and then all the eluates were collected; subsequently, 2 mL of acetonitrile was added for washing; the two eluates were combined and evaporated to dryness under nitrogen at 45°C; 1 mL of methanol was accurately added, the residue was vortexed to dissolve, and the residue was filtered through a 0.22 μm microporous membrane for HPLC-MS / MS online analysis.

6. The method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS according to claim 1, characterized in that: In the step 3.2), the ratio of acetonitrile to water is 85:

15.

7. The method for rapid determination of 17 new veterinary drug compounds in animal-derived foods by solid phase extraction combined with HPLC-MS / MS according to claim 1, characterized in that: When performing HPLC-MS / MS online analysis, The chromatographic conditions are: The chromatographic column Acquity UPLC HSS T3 (2.1 mm*100 mm, 1.8 μm, Waters Corporation, USA) was used for separation; the mobile phase A was methanol, and the mobile phase B was 0.2% formic acid water; the elution conditions of the veterinary drug residue mixture were: 5% A at 0–2 min, 5-90% A at 2–10 min, 90–95% A at 10–10.1 min, 95% A at 10.1–12 min, 95–5% A at 12–12.5 min, 5% A at 12.5–15 min; the total duration of the experiment was 15 min, the flow rate was 0.25 mL / min, and the injection volume was 2.0 μL; The mass spectrometry conditions were: The electrospray positive / negative ion (ESI+ / -) mode was adopted; the electrospray capillary voltage was set to 3.10 kV (ESI+) and 2.50 kV (ESI-), and the ESI cone voltage was set to 67 V (+) and 20 V (-), respectively; under the condition of a dissolution temperature of 450°C, the dissolved gas flow rates of ESI+ and ESI- were 800 L / h and 600 L / h, respectively, and the collision gas flow rates were both 0.15 mL / min.

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