A method for rapid determination of amprolium residues in animal foods based on one-step solid phase extraction

Abstract

The present invention discloses a method for rapidly determining the amount of amprolium residue in animal food based on one-step solid phase extraction. The sample is extracted with 80% acetonitrile solution (containing 0.2% formic acid), the supernatant is purified by one-step PRiME HLB solid phase extraction column, separated by BEH HILIC column, gradient elution is performed with acetonitrile-acidified ammonium formate as mobile phase, detection is performed in triple quadrupole mass spectrometry electrospray ionization multiple reaction monitoring mode, and quantitative analysis is performed by matrix matching external standard method. In the range of 0.25-100 ng / mL, the linear correlation coefficient R 2 The detection limit was 2.0 μg / kg and the quantification limit was 5.0 μg / kg. The spike recovery was 86.1%~108.5% and the relative standard deviation was 2.2%~8.4%. This method is simple and rapid in pretreatment and is suitable for the analysis and determination of amprolium residues in animal foods such as chicken, beef, liver, kidney, eggs and milk.

Description

Technical Field

[0001] The invention belongs to the technical field of food analysis, and particularly relates to a method for rapidly determining amprolium residues in animal food based on one-step solid phase extraction. Background Art

[0002] Amprolium, also known as Anbaoning, is an anti-thiamine anticoccidial drug with high efficiency and broad-spectrum bactericidal ability. It is widely used in the breeding process of many animals such as chickens, cattle, sheep, rabbits, etc. Because coccidiosis is contagious, it can significantly reduce the production efficiency of animals and even cause a large number of animal deaths, causing significant economic losses to breeders. In order to achieve the purpose of anticoccidial, there are unscientific and non-standard drug use in the breeding process, as well as non-strict implementation of drug withdrawal periods. The above factors can lead to amprolium residues in animal foods, which in turn affects the human body's absorption of thiamine, and may cause Wernicke's encephalopathy in severe cases. Therefore, many countries such as Japan, Canada, South Korea, and the United States have made clear provisions on the maximum residue limit of amprolium in animal foods. my country's GB 31650-2019 also clearly stipulates the maximum residue limit of amprolium in the muscle, liver, kidney, fat and eggs of cattle, chickens, turkeys. Therefore, it is of great significance to establish an analytical method for the residue of amprolium in animal foods and comprehensively evaluate the residue status of amprolium in foods to ensure consumer food safety.

[0003] The reported analytical methods for amprolium mainly include capillary electrophoresis, fluorescence spectroscopy, spectrophotometry, colorimetry, liquid chromatography and liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry combines the rapid and efficient separation capability of liquid chromatography with the qualitative and quantitative capabilities of mass spectrometry detectors. It has the characteristics of high sensitivity and strong anti-interference ability, and has become the main detection method for veterinary drug residues. Regarding the detection of amprolium, the current detection standards in my country mainly include "Determination of Amprolium Residues in Edible Tissues of Cattle" (GB 31613.1-2021), "Determination of Anticoccidial Drug Residues in Exported Animal Origin Foods by Liquid Chromatography-Mass Spectrometry / Mass Spectrometry" (SN / T 3144-2011) and "Determination of Amprolium Residues in Exported Animal Origin Foods by Liquid Chromatography-Mass Spectrometry / Mass Spectrometry" (SN / T 4583-2016). The pretreatment process of the three standards includes multiple steps such as target extraction, solvent conversion, solid phase extraction purification, and concentration. The process is complicated and time-consuming, and is not suitable for the rapid detection requirements of supervision. In addition, the GB31613.1 matrix is ​​incomplete and cannot meet the monitoring requirements of GB 31650. Therefore, it is urgent to establish a rapid and efficient detection method for amprolium in food to meet regulatory needs. Summary of the invention

[0004] This paper introduces a one-step solid phase extraction column purification for the first time, combined with a hydrophilic interaction chromatography column for separation and tandem mass spectrometry detection, and establishes a rapid detection method for amprolium residues in animal foods. The extraction solvent is optimized, and the acidified acetonitrile solution can achieve ionization of the target, weaken the interaction between the target and the solid phase extraction column, and ensure the recovery rate of the target; the purification process is optimized, and the PRiME HLB solid phase extraction column used can remove typical matrix interferences in animal foods such as proteins and phospholipids, and the purification process does not require activation and equilibration of the filler. The extraction solution can be directly loaded to obtain a clean sample solution, avoiding the steps of conventional SPE such as elution and washing, which greatly saves sample pretreatment time. At the same time, since the sample purification solution contains a high proportion of organic phase, it can be directly used for hydrophilic interaction chromatography injection analysis, and then the solvent conversion step is omitted again. Compared with the existing standard method, the experimental efficiency is significantly improved. Combined with the methodological validation results, this method has the characteristics of high sensitivity, good reproducibility, and convenient operation, which can provide technical support for the rapid detection of amprolium in animal foods.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for rapidly determining amprolium residues in animal food based on one-step solid phase extraction comprises the following steps:

[0007] (1) Sample extraction: weigh the sample, add the extractant, vortex mix, and ultrasonically centrifuge; add the extractant and repeat the extraction, combine the two extraction solutions, make up to volume, shake well and set aside;

[0008] (2) Sample purification: The extract is passed through a PRiME HLB solid phase extraction column, and the effluent is collected, vortexed and mixed, and then filtered through a microporous filter membrane for instrument measurement;

[0009] (3) The purified liquid of the sample to be tested is subjected to ultra-high performance liquid chromatography-tandem mass spectrometry detection.

[0010] Furthermore, the extractant is an 80% acetonitrile solution containing 0.2% formic acid.

[0011] Furthermore, the specific process of sample extraction was as follows: 2.5 g of sample was weighed into a 50 mL centrifuge tube, 20 mL of 80% acetonitrile aqueous solution containing 0.2% formic acid was added, vortexed for 1 min, ultrasonicated for 10 min, centrifuged at 8500 r / min for 2 min, and the supernatant was transferred to a 50 mL volumetric flask; 20 mL of extractant was added and the extraction was repeated once, the two extraction solutions were combined, and the volume was fixed to the mark with the extractant.

[0012] Furthermore, the specific process of sample purification is as follows: accurately pipette 3.0 mL of the above extract, inject it into the PRiME HLB solid phase extraction column, let the solution pass through the solid phase extraction column, and collect the effluent; drain the solid phase extraction column, vortex mix, and filter it through a 0.22 μm microporous filter membrane for instrument measurement.

[0013] Furthermore, the conditions of ultra-high performance liquid chromatography were as follows: chromatographic column: Waters ACQUITY UPLC BEH HILIC, size 100×2.1 mm, 1.7 μm; mobile phase: A is 5 mmol / L ammonium formate solution containing 0.05% formic acid, B is acetonitrile; flow rate: 0.30 mL / min; column temperature: 40 °C; injection volume: 2 μL; elution gradient:

[0014] .

[0015] Furthermore, the mass spectrometry conditions were as follows: electrospray ion source, positive ion scanning; detection mode: multiple reaction monitoring; electrospray voltage: 5500 V; nebulizer pressure: 55 L / h; curtain gas pressure: 45 L / h; auxiliary gas pressure: 50 L / h; collision gas pressure: 7 L / h; ion source temperature: 550 °C; auxiliary gas and nebulizer gas were compressed air, and collision gas and curtain gas were high-purity nitrogen; qualitative ion pairs, quantitative ion pairs, acquisition time, declustering voltage and collision energy were:

[0016] .

[0017] The beneficial effects of the present invention are:

[0018] The present invention forms a rapid determination method for amprolium in animal food based on one-step solid phase extraction. The method uses 80% acetonitrile solution (containing 0.2% formic acid) as an extractant. The high proportion of acetonitrile can denature the protein and clarify the filtrate, which provides a guarantee for the purification speed of SPE; at the same time, the presence of 0.2% formic acid also significantly improves the recovery rate of amprolium. The extract is purified in one step, without the need for solvent conversion before SPE, and also eliminates the five steps of activation, elution, elution, concentration of the eluent, and re-dissolution. Compared with the existing detection standards, the pretreatment efficiency is improved by 3-5 times. The method uses a BEH HILIC column for separation, with a moderate retention time, symmetrical chromatographic peaks, high response, and a sensitivity 2-5 times higher than the existing detection standards; the method has excellent recovery and reproducibility, and is suitable for the rapid detection of amprolium in batch samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of purification effects of different purification columns;

[0020] Figure 2is the recovery rate of different loading volumes;

[0021] Figure 3 The matrix effect of different loading volumes;

[0022] Figure 4 Characteristic ion chromatograms of 2.5 ng / mL bovine kidney standard solution (A), bovine kidney blank sample (B), and bovine kidney quantification limit spiked sample (C). DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the scheme of the present invention and to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the present invention is further described in detail below in conjunction with embodiments.

[0024] Example 1

[0025] 1 Experimental part

[0026] 1.1 Instruments, reagents and materials

[0027] AB SCIEX Triple Quad 6500+ ultra-high performance liquid chromatography-tandem mass spectrometer (equipped with electrospray ionization source) (AB SCIEX, USA); 3-18KS high-speed refrigerated centrifuge (Sigma, Germany); SECURA1102-1CN-SQP electronic balance (Sartorius Scientific Instruments Co., Ltd.); MS3 vortex mixer (IKA, Germany); DTC-33 ultrasonic cleaning machine (Hubei Dingtai Hengsheng Technology Equipment Co., Ltd.); Milli-Q ultrapure water system (Millipore, USA).

[0028] Acetonitrile (chromatographic grade, Honeywell Trading (Shanghai) Co., Ltd.), ammonium formate, formic acid (chromatographic grade, Sigma-Aldrich, USA); amprolium hydrochloride standard (CAS No.: 137-88-2, purity 98.6%) was purchased from Beijing Manhag Biotechnology Co., Ltd.; Oasis PRiME HLB solid phase extraction column (200 mg / 6mL, Waters, USA); BEH HILIC chromatographic column (100×2.1mm, 1.7 μm, Waters, USA).

[0029] Sample processing

[0030] 1.2.1 Extraction

[0031] Weigh 2.5 g of sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 20 mL of extractant (80% acetonitrile solution (containing 0.2% formic acid)), vortex mix for 1 min, ultrasonicate for 10 min, centrifuge at 8500 r / min for 2 min, and transfer the supernatant to a 50 mL volumetric flask. Add 20 mL of extractant and repeat the extraction once, combine the two extraction solutions, dilute to the mark with extractant, shake well and set aside.

[0032] 1.2.2 Purification

[0033] Accurately pipette 3.0 mL of the above extract and inject it into the PRiME HLB solid phase extraction column, so that the solution passes through the solid phase extraction column, collect the effluent, drain the solid phase extraction column, vortex mix, and filter through a 0.22 μm microporous membrane for instrument measurement.

[0034] Instrument analysis conditions

[0035] 1.3.1 Chromatographic conditions

[0036] a) Chromatographic column: Waters ACQUITY UPLC BEH HILIC (100×2.1mm, 1.7μm);

[0037] b) Mobile phase: A is 5 mmol / L ammonium formate solution containing 0.05% formic acid, B is acetonitrile, and the elution gradient is shown in Table 1;

[0038] c) Flow rate: 0.30 mL / min;

[0039] d) Column temperature: 40 °C;

[0040] e) Injection volume: 2 μL.

[0041] Table 1 UPLC gradient elution program

[0042]

[0043] 1.3.2 Mass spectrometry conditions

[0044] Electrospray ion source (ESI source), positive ion scanning; detection mode: multiple reaction monitoring (MRM); electrospray voltage: 5500 V; nebulizer pressure: 55 L / h; curtain gas pressure: 45 L / h; auxiliary gas pressure: 50 L / h; collision gas pressure: 7 L / h; ion source temperature: 550 ℃. The auxiliary gas and nebulizer gas are compressed air, and the collision gas and curtain gas are high-purity nitrogen. The qualitative ion pairs, quantitative ion pairs, acquisition time, declustering voltage and collision energy are shown in Table 2.

[0045] Table 2 Mass spectrometry parameters of amprolium

[0046]

[0047] *Quantitative ion

[0048] 2 Results and Discussion

[0049] 2.1 Optimization of pre-treatment methods

[0050] 2.1.1 Selection of extraction solvent

[0051] Amprolium is easily soluble in water, ethanol and methanol, but insoluble in chloroform. Phosphate, methanol, acetonitrile and trichloroacetic acid-acetonitrile solution are commonly used extraction solvents for the determination of amprolium. This method uses bovine kidney as a verification sample to investigate the extraction effects of five extraction solvents on amprolium: phosphate, methanol, acetonitrile, 80% acetonitrile water (containing 0.2% formic acid) and 1% trichloroacetic acid-acetonitrile (3:7). The experiment showed that the solution was turbid after phosphate extraction, while the other four extraction solutions were clear, and the recovery rates were all above 85%. This method intends to use the solid phase column pass-through mode for purification, and the extraction solvent is directly passed through the membrane and measured on the machine after passing through SPE, without a solvent conversion process. Pure methanol and pure acetonitrile have too strong elution ability for weak polar impurities such as phospholipids, affecting the purification effect; the trichloroacetic acid content in 1% trichloroacetic acid-acetonitrile (3:7) is relatively high, and it is directly fed into the mass spectrometer after passing through SPE, which has the risk of affecting the subsequent analysis of negative ion compounds. Therefore, the method preferentially selected 80% acetonitrile solution (containing 0.2% formic acid) as the extraction solvent.

[0052] This study also optimized the concentration of formic acid in the extraction solvent and verified the recovery of amprolium when formic acid accounted for 0%, 0.1%, 0.2%, 0.5% and 2% in 80% acetonitrile aqueous solution. The results showed that when no formic acid was added, the recovery rate of amprolium was 66.1%; when the formic acid concentration was 0.1%, the recovery rate was 82.3%; when the formic acid concentration was ≥0.2%, the recovery rate was higher than 90% and tended to be stable. The reason may be that when the amount of formic acid in the extraction solvent is insufficient, part of the amprolium is in a molecular state, and there is a reverse adsorption effect between it and SPE, so it is retained on SPE, resulting in a low recovery rate; when the formic acid concentration in the extraction solvent is ≥0.2%, all amprolium is in a cationic state, and the interaction between it and SPE is weak, and it cannot be retained on SPE, thereby improving the recovery rate of the method. In summary, 80% acetonitrile solution (containing 0.2% formic acid) was determined as the extraction solvent of this method.

[0053] 2.1.2 Selection of solid phase extraction columns

[0054] In order to reduce the influence of the matrix in animal food and reduce the contamination of mass spectrometry and chromatographic columns, this study investigated the purification of amprolium by three SPE methods: HLB, PRiME HLB and EMR-Lipid. HLB uses the target retention mode, while PRiME HLB and EMR-Lipid use the target pass-through mode. The experimental conditions are as follows:

[0055] (1) HLB solid phase extraction (60 mg, 3 mL): Extract according to step 1.2.1, accurately transfer 3 mL of sample extraction solution, blow nitrogen at 40°C until almost dry, re-dissolve with 5 mL of phosphate solution (pH = 6.0), and pass the re-solution through an HLB solid phase extraction column (pre-filtered with 3 mL of methanol, 3 mL of H2O). 2 O activation), and then 3 mL HO 2 O was used for elution, and finally 2 mL of methanol was added for elution. After the eluent was dried by nitrogen at 40°C, 1 mL of the initial mobile phase was accurately added for re-dissolution, vortexed for 3 min, and filtered through a 0.22 μm filter membrane for instrumental analysis.

[0056] (2) Solid-phase extraction: Extract according to step 1.2.1. Take 3 mL of sample extraction solution and inject it into PRiME HLB (200 mg, 6 mL) and EMR-Lipid (300 mg, 3 mL). Collect the sample purification solution in a 15 mL centrifuge tube, vortex mix, and filter through a 0.22 μm filter membrane for instrument measurement.

[0057] The effects of the three purification columns on recovery are detailed in Figure 1 .Depend on Figure 1 It can be seen that the recovery rates of HLB and PRiME HLB are both above 90%, and EMR-Lipid partially adsorbs amprolium with a recovery rate of 74.4%. PRiME HLB is an innovation based on HLB. Compared with HLB, it maintains good water wettability and has a stronger ability to remove proteins and phospholipids. The special sieve plate design, filler and manufacturing process can make the flow rate faster and more stable. This experiment selected PRiME HLB for one-step purification. This purification method does not require solvent conversion, and eliminates the activation, elution, and nitrogen concentration and re-dissolution of the eluent of SPE. The steps are simple and the inspection efficiency is higher.

[0058] This study further investigated the effects of three specifications of PRiME HLB solid phase extraction columns, 60 mg, 200 mg, and 500 mg, on recovery and matrix effect. The SPE recovery rate of 60 mg was 97.8%, the purification effect was poor due to less filler, and the matrix effect was 59.9%; the SPE recovery rate of 200 mg was 107.6%, and the matrix effect was 30.1%; the target was partially retained on the SPE with 500 mg filler, and the recovery rate was only 78.2%. After comprehensive consideration, this method finally selected the PRiME HLB (200 mg, 6 mL) solid phase extraction column for the purification of amprolium.

[0059] 2.1.3 Optimization of sample volume

[0060] For solid phase extraction, the sample volume will affect both the recovery rate and the purification effect. Therefore, this method compares the effects of sample volume on the recovery rate and matrix effect. The results are as follows: Figure 2 and Figure 3 shown. Figure 2 It showed that the recovery rate was slightly lower at 86.5% when the sample volume was 2 mL, and was stable at around 100% when the sample volume was ≥3 mL; Figure 3 The results showed that the larger the sample volume, the worse the purification effect and the more significant the matrix effect enhancement; the final sample volume was determined to be 3 mL.

[0061] Optimization of instrument analysis conditions

[0062] 2.2.1 Optimization of mass spectrometry conditions

[0063] Amprolium contains quaternary ammonium groups in its structure, which are very polar and suitable for electrospray ionization. Under acidic conditions, the amino groups in the amprolium molecular structure are easy to combine with protons to form positive ions, so the positive ion ionization mode is selected. 50 ng / mL of amprolium standard solution is loaded into the syringe pump, and a full scan is performed in the positive ion mode of the ESI source to obtain the molecular ion peak of amprolium [M-Cl+H] + The mass spectrometry parameters such as declustering voltage and collision energy were further optimized in the MRM mode. The optimal mass spectrometry conditions are shown in Table 1.

[0064] 2.2.2 Optimization of chromatographic conditions

[0065] 2.2.2.1 Selection of chromatographic columns

[0066] Amprolium is a quaternary amine compound and has weak retention in traditional reversed-phase columns. This study investigated three types of columns: HSS T3 (100 mm×2.1 mm, 1.8 μm), BEH Amide (100 mm×2.1 mm, 1.7 μm), and BEH HILIC (100 mm×2.1 mm, 1.7 μm). 18 Compared with BEH HILIC columns, it can provide stronger retention for polar compounds. Experiments show that when the proportion of acetonitrile is as low as 2%, the retention time of amprolium is 1.21min. At this time, the peaks of strong polar compounds in the sample to be tested are relatively concentrated, which is easy to interfere with the qualitative and quantitative analysis of the target. The remaining two hydrophilic columns can provide suitable chromatographic retention for amprolium. The peak shape on the BEH HILIC column is more symmetrical, and the response value is 2 times higher than that of BEH Amide. The BEH HILIC column uses unbonded ethylene bridge hybrid particles (BEH) particles. The filler is chemically stable in the pH range of 1-9. There is a hydrogen bond and dipole interaction between it and amprolium, which is very suitable for the determination of amprolium. The ZIC-HILIC column is a zwitterionic bonded phase with a sulfobetaine structure bonded to the surface of silica gel, and the suitable pH range is 3-8. When using SeQuant ZIC-HILIC (150 mm × 2.1mm, 5 μm) column to analyze amprolium, in addition to hydrogen bonding and dipole forces between amprolium and the stationary phase, there is also electrostatic interaction between amprolium and the charges on the ZIC-HILIC stationary phase. The interaction mechanism between ZIC-HILIC and the analyte is complex, the applicable pH range is narrow, and the method reproducibility is more challenging. Therefore, this method selects BEH HILIC column for the analysis of amprolium.

[0067] 2.2.2.2 Choice of mobile phase

[0068] Amprolium is easily ionized in positive ion mode, and acidic mobile phase can improve the mass spectrometric response of amprolium. In this study, six mobile phase combinations were compared, including 0.05% formic acid solution-acetonitrile, 5 mmol / L ammonium formate solution-acetonitrile, 5 mmol / L ammonium formate solution containing 0.05% formic acid-acetonitrile, 10 mmol / L ammonium formate solution containing 0.05% formic acid-acetonitrile, 5 mmol / L ammonium formate solution containing 0.1% formic acid-acetonitrile, and 5 mmol / L ammonium formate solution containing 0.2% formic acid-acetonitrile. The experiment showed that when acetonitrile-5mmol / L ammonium formate (containing 0.05% formic acid) was used as the mobile phase, the peak shape and response value of amprolium were the best, and the retention time was 3.22min. When the concentration of ammonium formate continued to increase to 10 mmol / L, the response value of amprolium decreased by 8 times; when the concentration of formic acid continued to increase in the acetonitrile-5 mmol / L ammonium formate system, the retention time and response value of the target did not change significantly. After comprehensive consideration, acetonitrile-5 mmol / L ammonium formate (containing 0.05% formic acid) was selected as the mobile phase.

[0069] Methodological investigation

[0070] 2.3.1 Linear range, detection limit and quantification limit

[0071] The matrix-matched standard working solutions (0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, 100 ng / mL) were subjected to UPLC-MS / MS analysis to obtain the mass chromatogram of the standard solution. The standard curve was drawn with the amprolium peak area (y) as the ordinate and the standard curve concentration (x) as the abscissa to obtain the linear regression equation. The linear regression equations and correlation coefficients of the 10 food matrices are shown in Table 3. The results showed that amprolium showed good linearity in the range of 0.25~100 ng / mL, and the correlation coefficient (R 2 ) are better than 0.99.

[0072] Table 3 Linear range, regression equation and correlation coefficient of amprolium in 10 food matrices

[0073]

[0074] A series of concentrations of standard substances were added to the above 10 blank samples, and the detection limit (LOD) of the method was determined at a signal-to-noise ratio of 3 times, and the quantification limit (LOQ) of the method was determined at a signal-to-noise ratio of 10 times. The detection limit and quantification limit of amprolium are 2.0 μg / kg and 5.0 μg / kg, respectively. The sensitivity of each matrix can meet regulatory requirements. The characteristic ion chromatograms of bovine kidney matrix-matched standard solution (2.5 ng / mL), bovine kidney blank sample, and bovine kidney quantification limit level sample are shown in Figure 2. Figure 4 shown.

[0075] 2.3.2 Recovery and precision

[0076] Ten blank matrices, including chicken, chicken liver, and milk, were selected for spike recovery tests at low, medium, and high levels (LOQ, 10LOQ, and 100LOQ). Each concentration level was measured six times in parallel to evaluate the recovery and precision of the method. The results are shown in Table 4. The results showed that the average recovery of amprolium in the ten matrices was 86.1-108.5%, with an RSD of 2.2-8.4%.

[0077] Table 4 Recovery and precision of amprolium in different matrices (n=6)

[0078]

[0079]

[0080] 2.4 Actual sample measurement

[0081] The method established in this paper was used to detect amprolium in a total of 56 batches of samples, including 10 batches of commercially available chicken, 5 batches of chicken liver, 5 batches of chicken kidney, 3 batches of chicken gizzard, 10 batches of eggs, 5 batches of beef, 5 batches of beef liver, 3 batches of beef kidney, 10 batches of milk and 1 batch of beef fat. Amprolium was detected in 2 batches of egg samples with detection values ​​of 56.6 μg / kg and 20.8 μg / kg, respectively. No other samples were detected.

[0082] Method accuracy test

[0083] This method and SN / T 4583-2016 were used to simultaneously test two batches of eggs with amprolium detected in 2.4 to verify the accuracy of this method. The test results are shown in Table 5. The results showed that the amprolium content in the two egg samples was basically consistent with the results of the standard method, with relative deviations of 6.19% and 9.57% respectively, indicating that this method has good accuracy. Since this method has simple operating steps, the parallelism of the test results is better.

[0084] Table 5 Comparison results between this method and the standard method

[0085]

[0086] 3 Conclusion

[0087] Based on one-step solid phase extraction, a rapid determination method for amprolium in animal food was developed. The method uses 80% acetonitrile solution (containing 0.2% formic acid) as the extractant. The high proportion of acetonitrile can denature the protein and clarify the filtrate, which provides a guarantee for the purification speed of SPE; at the same time, the presence of 0.2% formic acid significantly improves the recovery rate of amprolium. The extract is purified in one step, and no solvent conversion is required before SPE purification. It also eliminates the five steps of activation, elution, elution, concentration of the eluent, and re-dissolution. Compared with the existing detection standards, the pretreatment efficiency is increased by 3-5 times. This method uses a BEH HILIC column for separation, with moderate retention time, symmetrical chromatographic peaks, high response, and sensitivity 2-5 times higher than the existing detection standards; the recovery rate and reproducibility of the method are excellent, which is suitable for the rapid detection of amprolium in batch samples.

Claims

1. A method for rapidly determining amprolium residue in animal food based on one-step solid phase extraction, characterized in that: The following steps are involved: (1) Sample extraction: weigh the sample, add the extractant, vortex mix, and ultrasonically centrifuge; add the extractant and repeat the extraction, combine the two extraction solutions, make up to volume, shake well and set aside; (2) Sample purification: The extract is passed through a PRiME HLB solid phase extraction column, and the effluent is collected, vortexed and mixed, and then filtered through a microporous filter membrane for instrument measurement; (3) The purified liquid of the sample to be tested is subjected to ultra-high performance liquid chromatography-tandem mass spectrometry detection; The conditions for UHPLC were as follows: column: Waters ACQUITY UPLC BEH HILIC, dimensions 100 × 2.1 mm, 1.7 μm; Mobile phase: A is 5 mmol / L ammonium formate solution containing 0.05% formic acid, B is acetonitrile; flow rate: 0.30 mL / min; Column temperature: 40 ℃; injection volume: 2 μL; elution gradient: ； The mass spectrometry conditions were as follows: electrospray ion source, positive ion scanning; detection mode: multiple reaction monitoring; electrospray voltage: 5500 V; nebulizer pressure: 55 L / h; curtain gas pressure: 45 L / h; auxiliary gas pressure: 50 L / h; collision gas pressure: 7 L / h; ion source temperature: 550 °C; The auxiliary gas and nebulizer gas are compressed air, the collision gas and curtain gas are high-purity nitrogen; the qualitative ion pair, quantitative ion pair, acquisition time, declustering voltage and collision energy are: 。 2. The method for rapidly determining the amount of amprolium residue in animal food based on one-step solid phase extraction according to claim 1, characterized in that: The extractant was an 80% acetonitrile aqueous solution containing 0.2% formic acid.

3. The method for rapidly determining the amount of amprolium residue in animal food based on one-step solid phase extraction according to claim 1, characterized in that: The specific process of sample extraction was as follows: weigh 2.5 g of sample into a 50 mL centrifuge tube, add 20 mL of 80% acetonitrile solution containing 0.2% formic acid, vortex mix for 1 min, ultrasonicate for 10 min, centrifuge at 8500 r / min for 2 min, transfer the supernatant to a 50 mL volumetric flask; then add 20 mL of extractant and repeat the extraction once, combine the two extraction solutions, dilute to the mark with the extractant, shake well and set aside.

4. The method for rapidly determining the amount of amprolium residue in animal food based on one-step solid phase extraction according to claim 1, characterized in that: The specific process of sample purification is as follows: accurately pipette 3.0 mL of the above extract, inject it into the PRiMEHLB solid phase extraction column, let the solution pass through the solid phase extraction column, and collect the effluent; drain the solid phase extraction column, vortex mix, and filter it through a 0.22 μm microporous filter membrane for instrument measurement.

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