Method for detecting Hainanmycin residues in poultry meat, eggs and egg products and pretreatment method

Abstract

The invention provides a method for detecting Hainanmycin residues in poultry meat, eggs and egg products and a pretreatment method, and belongs to the technical field of food detection. The pretreatment method comprises the following steps: (1) adding a sample to be detected into an extraction solvent for extraction; (2) adding anhydrous magnesium sulfate and sodium chloride; (3) centrifuging to obtain supernate; (4) loading the supernate onto a solid-phase extraction small column, purifying and collecting to obtain first effluent; (5) loading an extraction solvent onto the small solid-phase extraction column, collecting to obtain a second effluent, and combining all the effluent; and (6) concentrating, redissolving and filtering all the effluent to obtain a sample solution to be detected. The invention further discloses a method for detecting Hainanmycin residues through liquid chromatography-mass spectrometry after a sample is pretreated, the method has the advantages of being rapid, efficient, low in detection limit and good in reproducibility, the blank of a method for detecting Hainanmycin in animal-derived food is filled, and the method is suitable for trace detection of Hainanmycin.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, and in particular relates to a method for detecting hainanmycin residues in poultry meat, eggs and egg products and a pretreatment method. Background Technology

[0002] Hainanmycin is a domestically developed polyether ionocarrier anticoccidial drug that has played a significant role in the prevention and control of coccidiosis in chickens since its introduction. However, toxicological studies have shown that Hainanmycin residues in animal-derived foods may pose a potential risk to consumer health. Given its importance, establishing a sensitive, accurate, and reliable detection method to monitor Hainanmycin residue levels in poultry meat, eggs, and egg products is of urgent practical significance for ensuring food safety.

[0003] Currently, research on detection methods for hainanmycin residues in animal-derived foods is incomplete, and there are gaps in the standard system. The existing detection methods mainly have the following problems: (1) Severe matrix interference and poor purification effect: The matrix of poultry meat (especially liver), poultry eggs and egg products (such as egg yolk powder and preserved eggs) is extremely complex, rich in fat, protein, phospholipids and endogenous pigments (such as carotenoids in egg yolks). These substances will produce severe ion inhibition or enhancement effects (matrix effect) in mass spectrometry detection and contaminate the chromatographic column and ion source. Existing pretreatment methods, such as simple liquid-liquid extraction (for example, using acetonitrile saturated n-hexane for degreasing), have a certain effect on removing proteins and some lipids, but have limited ability to remove small molecule polar interferences and pigments, resulting in high background interference in the final injection solution, making it difficult to guarantee detection sensitivity and accuracy. (2) Cumbersome pretreatment process or unstable recovery rate: Some solid phase extraction methods borrowed from other veterinary drug detection, such as the use of general solid phase extraction columns of hydrophilic-lipophilic balance (HLB) type, often require complex activation, rinsing and elution steps to balance the recovery rate and purification effect when dealing with polyether compounds with a specific structure like hainanmycin. The process is cumbersome and time-consuming, and hainanmycin may have a low recovery rate or large fluctuations in a strong hydrophilic or strong ion exchange environment due to poor retention behavior. In addition, traditional solid phase extraction methods have problems such as easy blockage of small columns and limited sample loading. (3) Lack of systematic solutions for poultry egg products: The few existing literature methods on hainanmycin are mostly focused on feed or animal tissues, and lack systematic pretreatment studies for egg products with different forms and compositions (such as liquid eggs, pickled and processed eggs, and dried egg powder). In particular, for egg products that have undergone processing and whose salt content and pH value have changed (such as preserved eggs and salted eggs), how to ensure the stable extraction and efficient purification of hainanmycin is still an unsolved technical problem.

[0004] Furthermore, existing purification methods for hainanmycin mostly employ liquid-liquid extraction or general-purpose solid-phase extraction columns (such as HLB). However, these methods have limited purification effects when dealing with poultry meat and egg products that are high in fat and pigments. Moreover, the retention behavior of hainanmycin on HLB columns is unstable, leading to large fluctuations in recovery rates and poor reproducibility. This is especially true for processed egg products (such as preserved eggs and salted eggs), whose high salt and high pH characteristics further increase the difficulty of purification. Therefore, developing a specific purification method that addresses the chemical characteristics of hainanmycin in poultry meat, eggs, and their complex egg product matrices, and is adaptable to various complex matrices, has become a pressing technical challenge in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for detecting hainanmycin residues in poultry meat, eggs, and egg products, along with a pretreatment method. This method is applicable to the determination of hainanmycin in poultry muscle, poultry liver, fresh poultry eggs, liquid egg products, dried egg products, frozen egg products, and processed eggs. The detection limit for hainanmycin is 0.2 μg / kg, and the quantitation limit is 0.5 μg / kg.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products, comprising the following steps: (1) Add acetonitrile to the sample to be tested for extraction; (2) Add anhydrous magnesium sulfate and sodium chloride to the mixture in step (1), shake and centrifuge to separate the supernatant; (3) Load the supernatant obtained in step (2) onto a neutral alumina solid-phase extraction column activated with acetonitrile and collect the first effluent; the neutral alumina solid-phase extraction column is used to specifically adsorb and remove lipids and pigment interferences in the sample matrix. (4) After the sample loading in step (3) is completed, the neutral alumina solid phase extraction column is washed with acetonitrile, and the second effluent is collected and combined with the first effluent in step (3); (5) Concentrate all the liquids combined in step (4) and redissolve them in acetonitrile, filter, and obtain the test solution.

[0007] Furthermore, in step (1), the extraction is performed under ultrasonic conditions, preferably with an ultrasonic frequency of 40 kHz and an extraction time of 15-20 min.

[0008] Further, in step (1), when the sample to be tested is poultry muscle, poultry liver, fresh egg, liquid egg product or frozen egg product, the ratio of sample to acetonitrile is 5g:(15-25)mL; when the sample to be tested is processed egg or dried egg product, water needs to be added to the sample to be tested for homogenization first, and then acetonitrile is added for extraction, wherein the ratio of processed egg to water is 5g:(8-12)mL, the ratio of processed egg to acetonitrile is 5g:(15-25)mL, the ratio of dried egg product to water is 1g:(2.5-3.5)mL, and the ratio of dried egg product to acetonitrile is 1g:(15-25)mL.

[0009] Furthermore, in step (2), the centrifugation speed is not less than 8000 r / min, and the centrifugation time is 8-10 min.

[0010] Furthermore, the mass ratio of the poultry muscle, poultry liver, fresh eggs, liquid egg products, frozen egg products, or processed egg samples to anhydrous magnesium sulfate and sodium chloride is 5:3:0.5; the mass ratio of the dried egg product samples to anhydrous magnesium sulfate and sodium chloride is 1:3:0.5.

[0011] Further, in step (3), the mass of the packing material of the neutral alumina solid-phase extraction column is 500 mg; when loading the sample, the mass ratio of the sample amount in the supernatant obtained from poultry muscle, poultry liver, fresh egg, liquid egg product, frozen egg product, and reconstituted egg sample to the mass of the packing material of the neutral alumina solid-phase extraction column is (1.75-2.25):1; the mass ratio of the sample amount in the supernatant obtained from dried egg product to the mass of the packing material of the neutral alumina solid-phase extraction column is (0.35-0.45):1. An anhydrous sodium sulfate layer with a height of 0.5 cm is pre-filled on the upper layer of the neutral alumina solid phase extraction column, and then activated with 5 mL of acetonitrile.

[0012] Furthermore, in step (5), the concentration is carried out by nitrogen blowing and the temperature is below 40°C; the pore size of the filter membrane used for filtration is 0.22μm.

[0013] This invention also provides a method for detecting hainanmycin residues in poultry meat, eggs, and egg products, comprising the following steps: The sample was processed using the above-mentioned sample pretreatment method to obtain the test solution, and the hainanmycin in the test solution was detected by high performance liquid chromatography-tandem mass spectrometry.

[0014] Furthermore, the detection conditions for the high-performance liquid chromatography are as follows: Column: ACQUITY UPLC BEH C18, 2.1×100mm, 1.7μm; Column temperature: 30℃; Mobile phase A: 5 mmol / L ammonium acetate aqueous solution (containing 0.1% (mass percentage, the same below) formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Flow rate: 0.5 mL / min; Injection volume: 5 μL; Elution method: gradient elution.

[0015] Furthermore, the gradient elution program is as follows: 0-0.5 min, 35% A; 0.5-4.0 min, 35% A-2% A; 4.0-10.0 min, 2% A; 10.1-13.0 min, 35% A.

[0016] Furthermore, the detection conditions for the tandem mass spectrometry are as follows: Quality analyzer: Triple quadrupole; Ion source: ESI+; Ion source spray voltage: 5500V; Ion source temperature: 400℃; Air curtain pressure: 30Psi; Atomizing gas pressure: 35Psi; Heating auxiliary gas pressure: 35Psi; Impact chamber emission voltage: 10V.

[0017] This invention uses neutral alumina solid-phase extraction (SPE) columns as the purification material. Unlike conventional reversed-phase or mixed-mode SPE materials, neutral alumina exhibits strong adsorption of lipids, pigments, and some polar impurities in the sample matrix, while retaining hainanmycin (a weakly polar polyether compound) relatively weakly. This allows for a balance between efficient purification and high recovery rates in a simple sample loading and washing process. This method overcomes the technical prejudice in the field that hainanmycin is unsuitable for SPE purification, providing a reliable pretreatment basis for trace detection of hainanmycin in poultry meat, eggs, and their products. Compared with existing technologies, this invention has the following advantages and technical effects: (1) In the sample pretreatment method of the present invention, acetonitrile is used to extract hainanmycin from the sample. Hainanmycin is a polyether substance, and polyether substances are mostly extracted with acetonitrile. Since hainanmycin has weak polarity and acetonitrile has a certain protein precipitation effect, the extraction efficiency of acetonitrile is relatively high. Therefore, acetonitrile is selected as the extraction solvent. When the sample to be tested is poultry muscle, poultry liver, fresh eggs, liquid egg products, frozen egg products, processed eggs, or dried egg products, anhydrous magnesium sulfate and sodium chloride are selected as dehydrating agent and salting-out agent, respectively, so that acetonitrile and aqueous phase are separated into layers, thereby maximizing the extraction efficiency of the compound to be tested. Anhydrous magnesium sulfate and sodium chloride solve the problem that if the amount of salting-out agent is too small, the salting-out effect will not be obvious. They also avoid the problem that if the amount is too large, some salting-out agent will enter the acetonitrile layer and suppress the mass spectrometry response signal.

[0018] (2) The sample pretreatment method of the present invention uses a neutral alumina solid-phase extraction column to purify the sample. The recovery rate of Hainanmycin is significantly better than that of hexane degreasing and general-purpose HLB solid-phase extraction column purification. The chromatographic peak after purification by neutral alumina solid-phase extraction column is free from impurity interference. The mass ratio of the solid-phase extraction column packing material to the mass of the sample to be tested in the supernatant is limited, which avoids the sample volume being too large and the solid-phase extraction column being overloaded, and also ensures the detection limit requirement of the compound to be tested.

[0019] (3) In the sample pretreatment method of the present invention, after the first effluent is collected, acetonitrile is loaded onto the solid phase extraction column to replace the purified liquid remaining in the solid phase extraction column, thereby reducing the loss of the target compound and improving the accuracy of the experiment. The concentration step adopts nitrogen blowing, which can not only concentrate multiple samples at the same time with high efficiency, but also nitrogen blowing is more suitable for a small number of samples than rotary evaporation, and avoids experimental errors caused by waste of samples during transfer. A filter membrane with a pore size of 0.22 μm is used for filtration to prevent damage to the chromatography caused by large-particle impurities in the sample, and also plays a role in purifying the sample to a certain extent.

[0020] (4) Based on the pretreatment of the sample in this invention, the residual amount of hainanmycin in the sample to be tested is detected by ultra-high performance liquid chromatography-mass spectrometry. This detection method has the characteristics of low interference, low detection limit, high sensitivity and good reproducibility, and is suitable for trace detection of veterinary drugs.

[0021] (5) This invention is the first to apply the neutral alumina solid-phase extraction technology system to the detection of hainanmycin residues, overcoming the long-standing technical prejudice in this field that solid-phase extraction has poor purification effects. By optimizing parameters such as sample loading and washing conditions, high recovery rates and low matrix effects were achieved in various poultry meat and egg product matrices, providing a reliable and universal detection method for the regulation of hainanmycin. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a flowchart illustrating the process for detecting hainanmycin residues in poultry meat, eggs, and egg products according to the present invention. Figure 2 The extraction ion chromatogram of the chicken liver-negative sample after adding hainanmycin in Example 1; Figure 3 The extraction ion chromatogram of the negative sample of preserved egg in Example 2 after adding hainanmycin; Figure 4 The extraction ion chromatogram of the egg yolk powder negative sample in Example 3 after adding hainanmycin; Figure 5 This is a comparison of the recovery rates of hainanmycin added in Comparative Example 1, Comparative Example 2, and Example 1. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention provides a pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products, comprising the following steps: (1) Add acetonitrile to the sample to be tested for extraction; (2) Add anhydrous magnesium sulfate and sodium chloride to the mixture in step (1), shake and centrifuge to separate the supernatant; (3) Load the supernatant obtained in step (2) onto a neutral alumina solid-phase extraction column activated with acetonitrile and collect the first eluent; the neutral alumina solid-phase extraction column is used to specifically adsorb and remove lipids and pigment interferences in the sample matrix. (4) After the sample loading in step (3) is completed, continue to wash the neutral alumina solid phase extraction column with acetonitrile, collect the second effluent and combine it with the first effluent in step (3); (5) Concentrate all the liquids combined in step (4) and redissolve them in acetonitrile, filter, and obtain the test solution.

[0029] In step (1) of the preferred embodiment of the present invention, the extraction is performed under ultrasonic conditions. Preferably, the ultrasonic frequency is 40 kHz and the extraction time is 15-20 min.

[0030] In step (1) of the preferred embodiment of the present invention, when the sample to be tested is poultry muscle, poultry liver, fresh egg, liquid egg product or frozen egg product, the ratio of sample to acetonitrile is 5g:(15-25)mL; when the sample to be tested is processed egg or dried egg product, water needs to be added to the sample to be tested for homogenization first, and then acetonitrile is added for extraction, wherein the ratio of processed egg to water is 5g:(8-12)mL, the ratio of processed egg to acetonitrile is 5g:(15-25)mL, the ratio of dried egg product to water is 1g:(2.5-3.5)mL, and the ratio of dried egg product to acetonitrile is 1g:(15-25)mL.

[0031] In step (2) of the preferred embodiment of the present invention, the centrifugation speed is not less than 8000 r / min and the centrifugation time is 8-10 min.

[0032] In a preferred embodiment of the present invention, the mass ratio of poultry muscle, poultry liver, fresh eggs, liquid egg products, frozen egg products, or processed egg samples to anhydrous magnesium sulfate and sodium chloride is 5:3:0.5; the mass ratio of dried egg product samples to anhydrous magnesium sulfate and sodium chloride is 1:3:0.5.

[0033] In step (3) of the preferred embodiment of the present invention, the mass of the packing material of the neutral alumina solid phase extraction column is 500 mg; when loading the sample, the mass ratio of the sample amount in the supernatant obtained from poultry muscle, poultry liver, fresh egg, liquid egg product, frozen egg product, and processed egg sample to the mass of the neutral alumina solid phase extraction column packing material is (1.75-2.25):1; the mass ratio of the sample amount in the supernatant obtained from dried egg product to the mass of the neutral alumina solid phase extraction column packing material is (0.35-0.45):1; an anhydrous sodium sulfate layer with a height of 0.5 cm is pre-filled on the packing material of the neutral alumina solid phase extraction column, and then activated with 5 mL of acetonitrile.

[0034] In step (5) of the preferred embodiment of the present invention, the concentration is carried out by nitrogen blowing and the temperature is below 40°C; the filter membrane used for filtration has a pore size of 0.22 μm.

[0035] This invention also provides a method for detecting hainanmycin residues in poultry meat, eggs, and egg products (see the process flow diagram for hainanmycin detection). Figure 1 ), including the following steps: The sample was processed using the above-mentioned sample pretreatment method to obtain the test solution, and the hainanmycin in the test solution was detected by high performance liquid chromatography-tandem mass spectrometry.

[0036] In a preferred embodiment of the present invention, the detection conditions for high-performance liquid chromatography are as follows: Column: ACQUITY UPLC BEH C18, 2.1×100mm, 1.7μm; Column temperature: 30℃; Mobile phase A: 5 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Flow rate: 0.5 mL / min; Injection volume: 5 μL; Elution method: gradient elution.

[0037] The gradient elution procedure for high performance liquid chromatography is shown in Table 1.

[0038] Table 1 Gradient elution program for high performance liquid chromatography In a preferred embodiment of the present invention, the detection conditions for tandem mass spectrometry are as follows: Quality analyzer: Triple quadrupole; Ion source: ESI+; Ion source spray voltage: 5500V; Ion source temperature: 400℃; Air curtain pressure: 30Psi; Atomizing gas pressure: 35Psi; Heating auxiliary gas pressure: 35Psi; Impact chamber emission voltage: 10V.

[0039] The information on the instruments and equipment used in the embodiments of this invention is as follows: Shimadzu LC-30A-AB QTrap 6500 Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometer (Shanghai Aibocaisi Analytical Instruments Trading Co., Ltd.); Nitrogen blowing device (Organomation, USA); Microporous filter membrane (0.22μm, Pall Corporation, USA); Ultrasonic cleaner (Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.); Vortex mixer (IKA GmbH, Germany); High-speed centrifuge (Sigma, Germany); ACQUITY UPLC BEH C18 2.1×100 mm, 1.7 μm column (Waters Corporation, USA). The standard for hainanmycin was purchased from the China Institute of Veterinary Drug Control. A 1000 mg / L standard stock solution was prepared with acetonitrile and stored at -20°C for no more than one year. Accurately measured amounts of the standard stock solution were prepared with acetonitrile to form a 10 μg / mL intermediate standard solution, which was also stored at -20°C for no more than six months.

[0040] Acetonitrile and formic acid (chromatographic grade) were purchased from Fisher Chemical Company, USA; ammonium acetate (chromatographic grade) was purchased from Beijing Bailingwei Technology Co., Ltd.; neutral alumina solid-phase extraction column (500 mg, 6 cc) was purchased from Waters Company; and experimental water was prepared using a Milli-Q ultrapure water system.

[0041] Hainanmycin standard solutions (all with a mass concentration of 100 ng / mL) were subjected to a full scan of the precursor ion in positive ion electrospray ionization mode to determine the quasi-molecular ion peak and obtain the corresponding [M+NH4] peak. + Peak. Secondary mass spectrometry was performed using the quasi-molecular ion peak as the parent ion. Three fragment ions with relatively high abundance were selected as quantitative and qualitative ions. The declustering voltage and collision energy of each ion pair were optimized, and the optimal conditions are shown in Table 2.

[0042] Table 2. MRM mass spectrometry parameters of hainanmycin A series of mixed standard solutions of different concentrations were prepared using extracts from chicken, duck, eggs, egg yolk powder, egg white, frozen eggs, and preserved eggs. These solutions were analyzed under the aforementioned conditions, and linear regression was performed on the peak area against the concentration. The results (Table 3) show that the linear relationship of hainanmycin is good, with a correlation coefficient (…). R 2 All are greater than 0.99.

[0043] Table 3. Linear equations, linear ranges, and correlation coefficients of hainanmycin in different matrix extraction solutions. The technical solution of the present invention will be further illustrated by the following embodiments.

[0044] Example 1 The chicken liver sample used in this embodiment was a negative sample of chicken liver with added hainanmycin standard at a dosage of 0.5 μg / kg. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to detect hainanmycin in the chicken liver, specifically including the following steps: a. Sample pretreatment: (1) Weigh 5g of chicken liver negative sample, add the working solution of hainanmycin to the sample, control the spiking amount to 0.5μg / kg, vortex to mix, let stand for 30min, then add 20mL of acetonitrile, vortex for 1min, extract under 40kHz ultrasound for 20min, add 3g of anhydrous magnesium sulfate and 0.5g of sodium chloride, shake and vortex for 2min, and centrifuge at 8000r / min for 10min.

[0045] (2) Take 4 mL of supernatant (containing 1 g of chicken liver negative sample) and load it onto a neutral alumina solid phase extraction column (the specification of the neutral alumina solid phase extraction column is 500 mg / 6 mL. Before use, add 0.5 cm of anhydrous sodium sulfate and activate it with 5 mL of acetonitrile). Maintain a flow rate of one drop per second and collect the first effluent. After the first effluent is collected, take another 4 mL of acetonitrile and load it onto the column to collect the second effluent. Combine all the effluents.

[0046] (3) The outflowing liquid nitrogen was concentrated to near dryness (temperature below 40℃), and the volume was adjusted to 1 mL with acetonitrile. The mixture was vortexed for 30 s and filtered through a 0.22 μm filter membrane to obtain the sample to be tested.

[0047] b. The presence of hainanmycin in the sample was detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry: The detection conditions for high-performance liquid chromatography are as follows: Column: ACQUITY UPLC BEH C18, 2.1×100mm, 1.7μm; Column temperature: 30℃; Mobile phase A: 5 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Flow rate: 0.5 mL / min; Injection volume: 5 μL; Elution method: gradient elution (see Table 1 for the specific gradient elution procedure); The detection conditions for tandem mass spectrometry are as follows: Quality analyzer: Triple quadrupole; Ion source: ESI+; Ion source spray voltage: 5500V; Ion source temperature: 400℃; Air curtain pressure: 30Psi; Atomizing gas pressure: 35Psi; Heating auxiliary gas pressure: 35Psi; Impact chamber emission voltage: 10V.

[0048] The extraction ion chromatogram of chicken liver-negative samples with added hainanmycin in Example 1 is shown below. Figure 2 This demonstrates that by using the method of the present invention for sample pretreatment and by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection, the residual amount of hainanmycin can be detected.

[0049] Example 2 The preserved egg samples used in this embodiment were negative samples of preserved eggs with added hainanmycin standard at a dosage of 0.5 μg / kg. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to detect hainanmycin in the preserved eggs, specifically including the following steps: a. Sample pretreatment: (1) Weigh 5g of the negative sample of preserved egg into a 50 mL centrifuge tube, add the working solution of Hainan mycin to the sample, control the spiking amount to 0.5 μg / kg, add 10 mL of water, vortex for 1 min, then add 20 mL of acetonitrile, vortex for 1 min, extract under 40 kHz ultrasound for 20 min, add 3g of anhydrous magnesium sulfate and 0.5g of sodium chloride respectively, shake and vortex for 2 min, and centrifuge at 8000 r / min for 10 min.

[0050] (2) Take 4 mL of supernatant (containing 1 g of the negative sample of preserved egg) and load it onto a neutral alumina solid phase extraction column (the specification of the neutral alumina solid phase extraction column is 500 mg / 6 mL. Before use, add 0.5 cm of anhydrous sodium sulfate and activate it with 5 mL of acetonitrile). Maintain a flow rate of one drop per second and collect the first effluent. After the first effluent is collected, take another 4 mL of acetonitrile and load it onto the column to collect the second effluent. Combine all the effluents.

[0051] (3) The outflowing liquid nitrogen was concentrated to near dryness (temperature below 40℃), and the volume was adjusted to 1 mL with acetonitrile. The mixture was vortexed for 30 s and filtered through a 0.22 μm filter membrane to obtain the sample to be tested.

[0052] b. The presence of hainanmycin in the sample was detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry: The detection conditions for high-performance liquid chromatography are as follows: Column: ACQUITY UPLC BEH C18, 2.1×100mm, 1.7μm; Column temperature: 30℃; Mobile phase A: 5 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Flow rate: 0.5 mL / min; Injection volume: 5 μL; Elution method: gradient elution (see Table 1 for the specific gradient elution procedure); The detection conditions for tandem mass spectrometry are as follows: Quality analyzer: Triple quadrupole; Ion source: ESI+; Ion source spray voltage: 5500V; Ion source temperature: 400℃; Air curtain pressure: 30Psi; Atomizing gas pressure: 35Psi; Heating auxiliary gas pressure: 35Psi; Impact chamber emission voltage: 10V.

[0053] The extraction ion chromatogram of the negative sample of preserved egg in Example 2 after the addition of hainanmycin is shown in the figure. Figure 3 This demonstrates that by using the method of the present invention for sample pretreatment and by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection, the residual amount of hainanmycin can be detected.

[0054] Example 3 The egg yolk powder sample used in this embodiment was a negative sample of egg yolk powder with added hainanmycin standard at a dosage of 0.5 μg / kg. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to detect hainanmycin in the egg yolk powder, specifically including the following steps: a. Sample pretreatment: (1) Weigh 1g of egg yolk powder negative sample into a 50 mL centrifuge tube, add the working solution of hainanmycin to the sample, control the spiking amount to 0.5 μg / kg, add 3 mL of water, vortex for 1 min, then add 20 mL of acetonitrile, vortex for 1 min, extract under 40 kHz ultrasound for 20 min, add 3g of anhydrous magnesium sulfate and 0.5g of sodium chloride respectively, shake and vortex for 2 min, and centrifuge at 8000 r / min for 10 min.

[0055] (2) Take 4 mL of supernatant (containing 0.2 g of negative sample of egg yolk powder) and load it onto a neutral alumina solid phase extraction column (the specification of the neutral alumina solid phase extraction column is 500 mg / 6 mL. Before use, add 0.5 cm of anhydrous sodium sulfate and activate it with 5 mL of acetonitrile). Maintain a flow rate of one drop per second and collect the first effluent. After the first effluent is collected, take another 4 mL of acetonitrile and load it onto the column to collect the second effluent. Combine all the effluents.

[0056] (3) The outflowing liquid nitrogen was concentrated to near dryness (temperature below 40℃), and the volume was adjusted to 1 mL with acetonitrile. The mixture was vortexed for 30 s and filtered through a 0.22 μm filter membrane to obtain the sample to be tested.

[0057] b. The presence of hainanmycin in the sample was detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry: The detection conditions for high-performance liquid chromatography are as follows: Column: ACQUITY UPLC BEH C18, 2.1×100mm, 1.7μm; Column temperature: 30℃; Mobile phase A: 5 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Flow rate: 0.5 mL / min; Injection volume: 5 μL; Elution method: gradient elution (see Table 1 for the specific gradient elution procedure); The detection conditions for tandem mass spectrometry are as follows: Quality analyzer: Triple quadrupole; Ion source: ESI+; Ion source spray voltage: 5500V; Ion source temperature: 400℃; Air curtain pressure: 30Psi; Atomizing gas pressure: 35Psi; Heating auxiliary gas pressure: 35Psi; Impact chamber emission voltage: 10V.

[0058] The extraction ion chromatogram of the negative sample of egg yolk powder in Example 3 after adding hainanmycin is shown in the figure below. Figure 4 This demonstrates that by using the method of the present invention for sample pretreatment and by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection, the residual amount of hainanmycin can be detected.

[0059] Recovery rate test Recovery tests were conducted by adding three different levels (low, medium, and high) of hainanmycin standard solution to blank chicken meat, duck meat, chicken liver, duck liver, chicken eggs, goose eggs, chicken egg yolk powder, duck egg white liquid, preserved eggs, and whole frozen goose eggs. Each addition level was tested in six parallel trials. The average recovery rate and relative standard deviation (RSD) are shown in Table 4. The recovery rate of hainanmycin ranged from 70% to 106%, with a precision (RSD) <12.4%, meeting the requirements for residue detection and exhibiting good reproducibility.

[0060] Table 4. Recovery range and relative standard deviation of hainanmycin in different matrices Comparative Example 1 The sample described in this comparative example was prepared by adding 0.5 μg / kg of hainanmycin standard to the chicken liver negative sample in Example 1. The hainanmycin in the sample was detected using the same ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) as in Example 1, with the only difference being the sample pretreatment method. The specific steps of the sample pretreatment method are as follows: (1) Weigh 5g of chicken liver negative sample, add the working solution of hainanmycin to the sample, control the spiking amount to 0.5μg / kg, vortex to mix, let stand for 30min, then add 20mL of acetonitrile, vortex for 1min, extract under 40kHz ultrasound for 20min, centrifuge at 8000r / min for 10min, concentrate the supernatant to near dryness (temperature below 40℃) by nitrogen blowing, and reconstitute with 2mL of water.

[0061] (2) Load all the reconstituted solution onto the activated HLB solid-phase extraction column (the specification of the HLB solid-phase extraction column is 500mg / 6mL, and it is activated with 5mL of methanol and 5mL of water in sequence before use). Maintain a flow rate of one drop per second, discard the first effluent, then load 4mL of water onto the column, discard the second effluent, and after the second effluent is drained, load 4mL of acetonitrile onto the column and collect the third effluent.

[0062] (3) The outflowing liquid nitrogen was concentrated to near dryness (temperature below 40℃), and the volume was adjusted to 1 mL with acetonitrile. The mixture was vortexed for 30 s and filtered through a 0.22 μm filter membrane to obtain the sample to be tested.

[0063] Comparative Example 2 The sample described in this comparative example was prepared by adding 0.5 μg / kg of hainanmycin standard to the chicken liver negative sample in Example 1. The hainanmycin in the sample was detected using the same ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) as in Example 1, with the only difference being the sample pretreatment method. The specific steps of the sample pretreatment method are as follows: (1) Weigh 5g of chicken liver negative sample, add the working solution of hainanmycin to the sample, control the spiking amount to 0.5μg / kg, vortex to mix, let stand for 30min, then add 10mL of acetonitrile, vortex for 1min, extract under 40kHz ultrasound for 20min, add 3g of anhydrous magnesium sulfate and 0.5g of sodium chloride, shake and vortex for 2min, and centrifuge at 8000r / min for 10min.

[0064] (2) Take 2 mL of supernatant and concentrate it to near dryness (temperature below 40℃) by nitrogen blowing. Make up to 1 mL with acetonitrile, add 3 mL of acetonitrile-saturated n-hexane (HEX), vortex for 2 min, and centrifuge at 8000 r / min for 10 min.

[0065] (3) Discard the acetonitrile-saturated n-hexane solution, take the lower layer of the complex solution, filter it through a 0.22 μm filter membrane, and obtain the sample to be tested.

[0066] The comparison results of the recovery rates of hainanmycin in Comparative Examples 1, 2 and Example 1 are shown in the figure. Figure 5 The recovery rate test method was the same as above. Compared with Example 1 (neutral alumina solid-phase extraction column, Al2O3), the recovery rate of hainanmycin in chicken liver measured using the pretreatment method (HLB) in Comparative Example 1 was significantly lower, with a recovery rate of only 58%. Therefore, the general-purpose HLB solid-phase extraction column sample purification pretreatment method is not only cumbersome but also leads to low recovery rates and inaccurate results. Compared with Example 1, the recovery rate of hainanmycin in chicken liver measured using the pretreatment method (HEX) in Comparative Example 2 was significantly lower. This is because hainanmycin has weak polarity, and some hainanmycin is lost after hexane extraction; the recovery rate of hainanmycin in Comparative Example 2 was only 67%. Therefore, the recovery rate of hainanmycin using hexane degreasing is low, and the results are inaccurate.

[0067] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products, characterized in that, Includes the following steps: (1) Add acetonitrile to the sample to be tested for extraction; the sample to be tested is poultry muscle, poultry liver, fresh eggs, liquid egg products, frozen egg products, processed eggs or dried egg products; (2) Add anhydrous magnesium sulfate and sodium chloride to the mixture obtained in step (1), shake and centrifuge to separate the supernatant; (3) Load the supernatant obtained in step (2) onto a neutral alumina solid-phase extraction column activated with acetonitrile and collect the first effluent; (4) After the sample loading in step (3) is completed, continue to wash the neutral alumina solid phase extraction column with acetonitrile, collect the second effluent and combine it with the first effluent in step (3); (5) Concentrate all the liquids combined in step (4) and redissolve them in acetonitrile, filter, and obtain the test solution.

2. The pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products according to claim 1, characterized in that, In step (1), when the sample to be tested is poultry muscle, poultry liver, fresh egg, liquid egg product or frozen egg product, the ratio of the sample to acetonitrile is 5g:(15-25)mL; when the sample to be tested is processed egg or dried egg product, water is first added to the sample to be tested for homogenization, and then acetonitrile is added for extraction, wherein the ratio of processed egg to water is 5g:(8-12)mL, the ratio of processed egg to acetonitrile is 5g:(15-25)mL, the ratio of dried egg product to water is 1g:(2.5-3.5)mL, and the ratio of dried egg product to acetonitrile is 1g:(15-25)mL.

3. The pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products according to claim 1, characterized in that, The mass ratio of the poultry muscle, poultry liver, fresh eggs, liquid egg products, frozen egg products, or processed egg samples to anhydrous magnesium sulfate and sodium chloride is 5:3:0.

5. The mass ratio of the dried egg product sample to anhydrous magnesium sulfate and sodium chloride was 1:3:0.

5.

4. The pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products according to claim 1, characterized in that, In step (3), the mass of the packing material in the neutral alumina solid-phase extraction column is 500 mg; When loading samples, the mass ratio of the sample amount in the supernatant obtained from poultry muscle, poultry liver, fresh eggs, liquid egg products, frozen egg products, or processed egg samples to the neutral alumina solid-phase extraction column packing is (1.75-2.25):1; the mass ratio of the sample amount in the supernatant obtained from dried egg products to the neutral alumina solid-phase extraction column packing is (0.35-0.45):

1.

5. The pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products according to claim 4, characterized in that, An anhydrous sodium sulfate layer with a height of 0.5 cm is pre-filled on the upper layer of the packing material of the neutral alumina solid phase extraction column.

6. The pretreatment method for detecting hainanmycin residues in poultry meat, eggs, and egg products according to claim 1, characterized in that, In step (5), the concentration is carried out by nitrogen blowing and the temperature is below 40°C; the filter membrane used for filtration has a pore size of 0.22 μm.

7. A method for detecting hainanmycin residues in poultry meat, eggs, and egg products, characterized in that, Includes the following steps: The sample is processed using the pretreatment method described in any one of claims 1-6 to obtain a test solution, and the hainanmycin in the test solution is detected by high performance liquid chromatography-tandem mass spectrometry.

8. The method according to claim 7, characterized in that, The detection conditions for the high-performance liquid chromatography are as follows: Column: ACQUITY UPLC BEH C18, 2.1×100mm, 1.7μm; Column temperature: 30℃; Mobile phase A: 5 mmol / L ammonium acetate aqueous solution containing 0.1 wt% formic acid; Mobile phase B: acetonitrile containing 0.1 wt% formic acid; Flow rate: 0.5 mL / min; Injection volume: 5 μL; Elution method: gradient elution.

9. The method according to claim 8, characterized in that, The gradient elution program is as follows: 0-0.5 min, 35% A; 0.5-4.0 min, 35% A-2% A; 4.0-10.0 min, 2% A; 10.1-13.0 min, 35% A.

10. The method according to claim 7, characterized in that, The detection conditions for the tandem mass spectrometry method are as follows: Quality analyzer: triple quadrupole; Ion source: ESI+; Ion source spray voltage: 5500V; Ion source temperature: 400℃; Air curtain pressure: 30Psi; Atomizing gas pressure: 35Psi; Heating auxiliary gas pressure: 35Psi; Impact chamber emission voltage: 10V.

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