CN113702526A - Method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs - Google Patents

Method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs Download PDF

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CN113702526A
CN113702526A CN202110972164.0A CN202110972164A CN113702526A CN 113702526 A CN113702526 A CN 113702526A CN 202110972164 A CN202110972164 A CN 202110972164A CN 113702526 A CN113702526 A CN 113702526A
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albendazole
poultry
pork
eggs
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谢恺舟
姜军华
刁志祥
谢星
庞茂达
郭亚文
陈兰
陈晋元
贺兆源
卢阳
王波
张跟喜
张涛
戴国俊
王冉
卜仕金
包文斌
高玉时
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Yangzhou University
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Abstract

The invention relates to the field of veterinary drug residue detection, and particularly relates to an analysis method for efficiently and simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs. Sequentially extracting pork, poultry meat and poultry eggs with an extractant, degreasing with n-hexane, purifying an extracting solution with an HLB solid phase extraction column, blowing the purified sample solution into a nitrogen blowing instrument with nitrogen for drying, and detecting by using a UPLC-FLD system after redissolution; the extracting agent adopted by the pork and the poultry meat is ethyl acetate, and the extracting agent adopted by the poultry eggs is formic acid-acetonitrile; the albendazole and three metabolites thereof are albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-amino sulfone. The method is rapid, efficient, accurate in quantification, high in recovery rate and sensitivity, and capable of meeting relevant standards for veterinary drug residue detection in international trade and meeting requirements for veterinary drug residue detection.

Description

Method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs
Technical Field
The invention relates to the field of veterinary drug residue detection, and particularly relates to an analysis method for efficiently and simultaneously detecting albendazole and multi-residues of three metabolites (albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone) of the albendazole in pork, poultry and eggs.
Background
Methods for simultaneously detecting albendazole and three metabolites thereof (albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone) in animal derived food at home and abroad mainly comprise an immunoassay method, a gas chromatography method, a high performance liquid chromatography method, a liquid chromatography-mass spectrometry combined method and the like, but a method for simultaneously detecting albendazole and residues of the three metabolites thereof in pork, poultry meat (chicken, duck meat and goose meat) and poultry eggs (eggs, duck eggs and goose eggs) by using an ultra high performance liquid chromatography-fluorescence detection method (UPLC-FLD) is not reported at home and abroad. Therefore, the research establishes a UPLC-FLD method for simultaneously detecting the multi-residues of albendazole and three metabolites thereof (albendazole sulfone, albendazole sulfoxide and albendazole-2-amino sulfone) in pork, poultry meat (chicken, duck meat and goose meat) and eggs (eggs, duck eggs and goose eggs), and provides a new detection method and technical support for simultaneously detecting the multi-residues of the albendazole and the three metabolites thereof in the pork, the poultry meat and the eggs.
Disclosure of Invention
In order to quickly, efficiently and accurately detect Albendazole (ABZ) and three metabolites of albendazole sulfone (ABZ-SO) in pork, poultry meat (chicken, duck meat and goose meat) and poultry eggs (eggs, duck eggs and goose eggs) simultaneously2) Albendazole sulfoxide (ABZ-SO) and albendazole-2-aminosulfone (ABZ-2 NH)2-SO2) And residue, the invention provides an ultra-high performance liquid chromatography-fluorescence detection method. The method is rapid, efficient, accurate in quantification, high in recovery rate and sensitivity, and capable of meeting relevant standards for veterinary drug residue detection in international trade and meeting requirements for veterinary drug residue detection.
The technical scheme provided by the invention is as follows:
a method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs comprises the following steps:
sequentially extracting pork, poultry meat and poultry eggs with an extractant, degreasing with n-hexane, purifying an extracting solution with an HLB solid phase extraction column, blowing the purified sample solution into a nitrogen blowing instrument with nitrogen for drying, and detecting by using a UPLC-FLD system after redissolution; the extracting agent adopted by the pork and the poultry meat is ethyl acetate, and the extracting agent adopted by the poultry eggs is formic acid-acetonitrile; the albendazole and three metabolites thereof are albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-amino sulfone.
Further, the solid phase extraction process comprises the following steps: waters
Figure BDA0003226070510000022
Sequentially activating and balancing a PRIME HLB solid-phase extraction column by using methanol and ultrapure water; leaching the sample solution with a mobile phase, draining the extraction column, and eluting with ammoniated methanol; the ammoniated methanol is ammonia water, methanol, V/V, 2: 8.
Further, extracting pork, poultry meat and eggs with an extracting agent in sequence, performing vortex oscillation, performing ultrasonic-assisted extraction for 5-10 min, centrifuging for 5-10 min, transferring supernate after centrifugation is finished, repeating the extraction steps on residues, combining the supernate obtained in two times, and drying with nitrogen.
Further, with ACQUITY
Figure BDA0003226070510000023
BEH C18The chromatographic column serves as an analytical column.
Further, the mobile phase A is acetonitrile; the mobile phase B is 0.2% formic acid water solution, and the mobile phase B contains 0.05% triethylamine.
Further, the fluorescence detector adopts a single-channel detection mode, and the wavelengths are set as follows: an excitation wavelength of 286nm and an emission wavelength of 335 nm.
Further, the column temperature is 35 ℃, and the flow rate is 0.25 mL/min; the injection volume was 5. mu.L.
Further, the elution mode adopts isocratic elution, and A: B: 31: 69 (V/V).
Further, the poultry egg extraction agent is 0.2% formic acid-acetonitrile.
Further, the detection formulas of pork, poultry meat and poultry eggs are respectively as follows:
Figure BDA0003226070510000021
a method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs comprises the following steps:
accurately weighing (2.00 +/-0.02) g of homogenized pork, poultry meat and egg samples into a 50mL polypropylene centrifuge tube, respectively adding an extracting agent (15 mL of ethyl acetate for pork and poultry meat and 10mL of 0.2% formic acid-acetonitrile for poultry eggs), performing ultrasonic-assisted extraction for 5min after vortex oscillation, then centrifuging for 10min, transferring the supernatant into the 50mL polypropylene centrifuge tube after centrifugation is finished, repeating the extraction steps on residues, and combining the two supernatants for later use. Blowing nitrogen to near dryness, dissolving residue with 5mL mobile phase, defatting with 15mL acetonitrile saturated n-hexane, vortex vibrating for 2min, centrifuging at 6000 Xg for 5min, discarding n-hexane layer, and passing through Waters
Figure BDA0003226070510000031
And (4) purifying the PRIME HLB solid-phase extraction column. Blowing the purified sample liquid in a nitrogen blowing instrument by using nitrogen for drying, re-dissolving the dried sample by using 2mL of mobile phase, carrying out low-speed vortex for 1min, absorbing by using a sterile syringe, passing through a 0.22 mu m hydrophilic PTFE type needle filter, and detecting the filtrate by using an UPLC-FLD system.
Advantageous effects
The invention utilizes ACQUITY
Figure BDA0003226070510000032
BEH C18A chromatographic column (2.1mm × 100mm, 1.7 μm) is used as an analysis column, acetonitrile-formic acid aqueous solution (containing 0.05% triethylamine) is used as a mobile phase, an elution mode adopts a mode of isocratic elution (31: 69) to separate four targets, the flow rate is 0.25mL/min, the column temperature is 35 ℃, the sample injection volume is 5 μ L, a fluorescence detector adopts a single-channel detection mode, and the wavelength is set as follows: the excitation wavelength is 286nm, the emission wavelength is 335nm, and the target substances of the albendazole, the albendazole sulfone, the albendazole sulfoxide and the albendazole-2-aminosulfone are separated from impurities, are not interfered with each other, have no trailing peak and leading edge, and have better separation degree.
The invention provides an ultra-high performance liquid chromatography fluorescence detection (UPLC-FLD) method for simultaneously detecting albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-amino sulfone multi-residues in pork, poultry (chicken, duck and goose) and poultry eggs (eggs, duck eggs and goose eggs), which is rapid and efficient, high in sensitivity and good in recovery rate, can provide a new technical support for simultaneously detecting albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-amino sulfone multi-residues in animal-derived tissues, and meets the relevant standards for veterinary drug residue detection in international trade.
Drawings
FIG. 1 chromatogram of standards (A), blank pork (B) and blank pork plus standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 2 chromatogram of standard (A), blank chicken muscle (B) and blank chicken muscle add standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 3 chromatograms of standard (A), duck muscle blank (B) and duck muscle addition standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 4 chromatograms of standard (A), white goose muscle (B) and white goose muscle addition standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 5 chromatograms of standard (A), hen egg blank (B) and hen egg add standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 6 chromatograms of standard (A), blank duck egg (B) and blank duck egg additive standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 7 chromatograms of standard (A), blank goose eggs (B) and blank goose egg additive standard (C) (albendazole: 50. mu.g/kg, albendazole sulfone: 10. mu.g/kg, albendazole sulfoxide: 50. mu.g/kg albendazole-2-aminosulfone: 10. mu.g/kg);
FIG. 8 illustrates standard curves for substrate addition of albendazole-2-aminosulfone (A), albendazole sulfoxide (B), albendazole sulfone (C), and albendazole (D) in pork and poultry meat;
FIG. 9 shows standard curves for matrix addition of albendazole-2-aminosulfone (A), albendazole sulfoxide (B), albendazole sulfone (C), and albendazole (D) in egg.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The source, trade name and composition of the reagents used are indicated at the first appearance and the same reagents used thereafter are the same as indicated for the first time unless otherwise specified.
(I) raising and sample collection of experimental livestock and poultry
Respectively randomly selecting 8 ternary hybrid pigs (Changzhou Kangle farming and animal husbandry Co., Ltd.) with the age of 180 days, 10 Yangzhou yellow chickens (Jiangsu Jinghai poultry industry Co., Ltd.) with the age of 70 days, 10 Gaoyou ducks (Jiangsu Gaoyou duck group Co., Ltd.) with the age of 140 days, 10 Yangzhou gooses (Yangzhou Song goose industry development Co., Ltd.) with the age of 70 days, breeding the male and female half pigs in a single cage (ring) in a test, freely drinking water, and feeding the complete feed (Yangzhou Yang City large feed factory) without any medicine. Slaughtering after 15 days, taking test pig hip muscle and poultry breast muscle as blank muscle samples, respectively, pulverizing with a knife grinder, vacuumizing, subpackaging, respectively labeling, sealing, and storing in a refrigerator at-34 deg.C for use.
Respectively selecting 35 egg-laying Jinghai yellow chickens (Jiangsu Jinghai poultry group limited company) with the age of 31 weeks, 35 egg-laying Gaoyou ducks (Jiangsu Gaoyou duck group limited company) with the age of 28 weeks and 50 egg-laying Yangzhou geese (Yangzhou Tiange goose industry development limited company) with the age of 32 weeks, feeding the chickens in a single cage for 15 days before the test, freely drinking water, and feeding the chickens with full-value feed (Yangzhou Yang feed factory) without any medicine. Eggs were collected 16:00-17:00 pm on day 16, and were collected for two consecutive weeks. The poultry egg sample is collected, homogenized, subpackaged, labeled, sealed and stored in a refrigerator at the temperature of minus 34 ℃ for later use.
(II) the extraction and purification steps of the invention
1. Taking out the blank sample from a refrigerator, thawing to room temperature, accurately weighing (2.00 +/-0.02) g of the homogenized blank muscle sample into a 50mL polypropylene centrifuge tube, respectively adding an extracting agent (15 mL of ethyl acetate for pork and poultry meat and 10mL of 0.2% formic acid-acetonitrile for poultry eggs), carrying out vortex oscillation for 5min, carrying out ultrasonic-assisted extraction for 5min, carrying out refrigerated centrifugation for 10min at 4 ℃ and 12000 Xg, transferring the supernatant into the 50mL polypropylene centrifuge tube after the centrifugation is finished, repeating the extraction steps on residues, and combining the two supernatants for later use;
2. blowing nitrogen to near dryness, dissolving the residue with 5mL mobile phase, adding 15mL acetonitrile saturated n-hexane for degreasing, vortex shaking for 2min, centrifuging at 6000 Xg for 5min, and discarding the n-hexane layer. Solid phase extraction process: waters
Figure BDA0003226070510000051
Sequentially activating and balancing a PRIME HLB solid-phase extraction column by using 3mL of methanol and 3mL of ultrapure water; leaching the sample solution with 3mL of mobile phase, draining the extraction column, and eluting with 3mL of ammoniated methanol (ammonia water: methanol: 2:8, V/V) in a 10mL glass centrifuge tube for later use;
3. and blowing the purified sample liquid to be nearly dry in a nitrogen blowing instrument by using nitrogen (blowing the nitrogen blowing pipeline for 3min before starting blowing to prevent impurity gas from remaining in the gas pipeline, finely adjusting the flow control valve to a proper flow rate to enable the gas flow to generate ripples on the surface of the sample and not splash, and continuously adjusting the position of a needle head in the nitrogen blowing process). The dried sample was redissolved with 2mL of mobile phase, vortexed at low speed for 1min, pipetted using a sterile syringe, passed through a 0.22 μm hydrophilic PTFE-style syringe filter, and the filtrate was tested on a UPLC-FLD system.
(III) ultra-high performance liquid chromatography conditions
A chromatographic column: ACQUITY
Figure BDA0003226070510000061
BEH C18 column (2.1 mm. times.100 mm, 1.7 μm); pre-column: VanGuardTM Pre-Column C18 Column (2.1 mm. times.5 mm, 1.7 μm); column temperature: 35 ℃; mobile phase A: acetonitrile; mobile phase B: 0.2% aqueous formic acid (containing 0.05% triethylamine); isocratic elution A: B: 31: 69 (V/V); a detector: a fluorescence detector (excitation wavelength 286nm, emission wavelength 335 nm); flow rate: 0.25 mL/min; sample introduction volume: 5 μ L.
(IV) quantitative method
1. Determination of detection and quantitation limits
Diluting the low-concentration albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone standard working solution by blank matrix extracting solution step by step, detecting by UPLC-FLD, measuring each concentration for 3 times, and obtaining the average value of signal-to-noise ratio (S/N). When the S/N is more than or equal to 3, the corresponding addition concentration is the detection Limit (LOD) of the analysis method; when the S/N is more than or equal to 10, the corresponding addition concentration is the quantitative Limit (LOQ) of the analysis method.
The results show that: the LOD of albendazole in pork, poultry meat (chicken, duck and goose muscles) and eggs (whole chicken, duck and goose eggs) is 2.8, 3.2, 3.1, 3.6, 3.8, 3.6 and 3.9 mug/kg, and the LOQ is 10.0, 10.7, 10.0, 10.9, 12.6, 11.5 and 12.3 mug/kg; albendazole sulfone has an LOD of 0.4, 0.3, 0.2, 0.4, 0.5 and 0.7. mu.g/kg and an LOQ of 1.5, 1.0, 1.5, 1.7 and 1.9. mu.g/kg; the LOD of albendazole sulfoxide was 3.8, 3.0, 2.4, 2.5, 3.1, 2.8 and 2.9 μ g/kg, and the LOQ was 9.7, 8.3, 8.0, 8.5, 9.3, 9.6 and 9.8 μ g/kg; albendazole-2-aminosulfone has an LOD of 0.9, 0.6, 0.5, 0.7, 1.1 and 0.8. mu.g/kg and an LOQ of 3.0, 1.8, 1.5, 1.9, 2.3, 2.8 and 2.6. mu.g/kg. The above research results show that: the detection limit and the quantitative limit of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone in pork, poultry meat (chicken, duck and goose muscles) and poultry eggs (whole chicken, duck and goose eggs) are lower, and the sensitivity is high.
2. Drawing of standard curve
Accurately measuring appropriate amounts of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone standard working solutions with appropriate concentrations, diluting with acetonitrile: 0.2% formic acid aqueous solution (containing 0.05% triethylamine): 31: 69(V/V) solution to obtain a series of mixed standard working solutions (corresponding concentrations of albendazole in pork and poultry meat tests are 10.0, 20.0, 25.0, 50.0, 100.0, 200.0 and 400.0 μ g/L, corresponding concentrations of albendazole sulfone are 1.0, 10.0, 25.0, 50.0, 100.0, 200.0 and 400.0 μ g/L, corresponding concentrations of albendazole sulfoxide are 8.0, 10.0, 25.0, 50.0, 100.0, 200.0 and 400.0 μ g/L, corresponding concentrations of albendazole-2-aminosulfone, 1.5, 0.0, 10.0 and 11.0 μ g/L, respectively, and corresponding concentrations of albendazole in poultry egg tests are 1.0, 10.0, 0 μ g/L, and 1.0, 0.0, 0 μ g/L, and 11.0 μ g/L, respectively, 20.0, 25.0, 50.0, 100.0, 200.0 and 400.0 mu g/L of albendazole sulfone with corresponding concentration of 1.0, 10.0, 25.0, 50.0, 100.0, 200.0 and 400.0 mu g/L respectively, albendazole sulfoxide with corresponding concentration of 9.0, 10.0, 25.0, 50.0, 100.0, 200.0 and 400.0 mu g/L respectively, albendazole-2-aminosulfone with corresponding concentration of 2.0, 10.0, 25.0, 50.0, 100.0, 200.0 and 400.0 mu g/L respectively, sample injection is carried out in sequence from low concentration to high concentration, measurement is carried out in sequence under optimized UPLC-FLD condition, each concentration is measured for 5 times, the average chromatographic concentration of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone is respectively plotted as a horizontal coordinate, and a standard horizontal coordinate is plotted as a standard.
As can be seen from FIGS. 8 and 9, the peak areas of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone showed good linear relationship with the added concentration, the linear range, the regression equation and the determination coefficient R2See table 1. If the concentration analyzed exceeds the linear range of the sample, the sample is diluted to be in the linear range accurately, and the detected result is multiplied by the corresponding multiple to be converted.
TABLE 1 Linear ranges, regression equations and determinant coefficients for albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone in pork, poultry and poultry eggs
Figure BDA0003226070510000071
3 calculation of recovery and precision
Accurately weighing (2.00 +/-0.02) g of well-homogenized blank pork, poultry meat and poultry egg samples, respectively adding a proper amount of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone mixed standard working solution (because the LOQ of various medicines in pork, poultry meat and poultry eggs are different, the LOQ concentration point in each sample is respectively added by adopting the standard working solution with the medicine concentration of 0.1 mu g/mL), so that the adding concentrations of various medicines in corresponding sample matrixes are respectively LOQ, 0.5MRL, 1.0MRL and 2.0MRL, each concentration is set to be 6 groups of parallel, then carrying out on-machine detection on the filtrate after the samples in the step (II) are extracted and purified, substituting the measured average peak area of each medicine into a standard curve, and calculating the adding recovery rate.
Chromatogram of standard (A), blank pork (B) and blank pork added with standard (C) (albendazole-2-amino sulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, albendazole: 50 μ g/kg) are shown in figure 1; chromatogram of standard (A), blank chicken muscle (B) and blank chicken muscle adding standard (C) (albendazole-2-amino sulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, albendazole: 50 μ g/kg) are shown in FIG. 2; chromatogram of standard (A), blank duck muscle (B) and blank duck muscle adding standard (C) (albendazole-2-amino sulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, albendazole: 50 μ g/kg) are shown in FIG. 3; chromatogram of standard (A), blank goose muscle (B) and blank goose muscle addition standard (C) (albendazole-2-amino sulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, albendazole: 50 μ g/kg) are shown in FIG. 4; chromatograms of standard substance (A), blank egg (B) and blank egg with standard substance (C) (albendazole-2-aminosulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, albendazole: 50 μ g/kg) are shown in FIG. 5; chromatograms of standard (A), blank duck egg (B) and blank duck egg with standard (C) (albendazole-2-aminosulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, and albendazole: 50 μ g/kg) are shown in FIG. 6; chromatograms of standard (A), blank goose egg (B) and blank goose egg with standard (C) (albendazole-2-aminosulfone: 10 μ g/kg, albendazole sulfoxide: 50 μ g/kg, albendazole sulfone: 10 μ g/kg, and albendazole: 50 μ g/kg) are shown in FIG. 7. As can be seen from fig. 1 to 7, the UPLC-FLD method for simultaneously detecting multiple residues of albendazole and three metabolites thereof (albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone) in pork, poultry (chicken, duck, goose) and poultry eggs (eggs, duck eggs and goose) established in the research has the advantages of isocratic elution, stable baseline, no drift, good chromatographic peak shape, no interference of impurity peaks, and only 6min of detection time, and compared with the HPLC-FLD method, the method disclosed by the invention is rapid and efficient, and greatly improves the working efficiency.
Precision within day (batch): at different time points on the same day, respectively carrying out 6 times of parallel measurement on 4 samples with added concentration by using the same instrument and the same standard curve, and calculating the precision within the day; daytime (batch) precision: on different days of the week, the day precision was calculated by performing 6 replicates of each of the 4 concentrations of the sample using the same instrument using different standard curves (standard curves were re-plotted daily).
Under the condition, the adding recovery rate and the precision of the albendazole, the albendazole sulfone, the albendazole sulfoxide and the albendazole-2-amino sulfone extracted from the pork, the poultry meat and the poultry eggs by the method are shown in tables 2 to 8. As can be seen from tables 2 to 8, the addition recovery rates of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone in pork, poultry meat and poultry eggs are high, the accuracy and the repeatability are good, and the precision is high.
(V) optimization of sample pretreatment process and chromatographic conditions
1. Selection of extraction solvent
Albendazole and three metabolite molecules thereof belong to medium-polarity molecules and are weakly alkaline, and the selection of an extracting agent needs to take the extraction capacity of the drug and the removal capacity of interfering substances in a sample matrix into consideration. This experiment compares the extraction effect of using ethyl acetate, acetonitrile and acetonitrile-0.2% acetic acid as the extractant. The results show that: although the extraction effect of acetonitrile is good, due to the fact that the polarity of acetonitrile is large, more endogenous substances are extracted from pork and poultry meat, and impurity peaks interfere with target substance chromatographic peaks during on-machine detection. And the ethyl acetate is low in toxicity and volatile, so that the time can be saved during concentration, the test progress is accelerated, and the detection efficiency is improved. When the ethyl acetate is used as the extractant, the target substance can be effectively extracted from the sample matrix, the extracted impurities are few, the interference is small, and the obtained recovery rate is over 80 percent, so the ethyl acetate is used as the extractant in the experiment. In addition, in order to improve the recovery rate, the test inspects the extraction times of 1, 2 and 3 times, and the result shows that the recovery rate can be effectively improved by two times of extraction compared with one time of extraction, the improvement of the recovery rate by three times of extraction is not obvious, and the waste amount of the solvent is large, so that the test is fully considered from the aspects of recovery rate and environmental protection, and the final sample pretreatment process is two times of extraction.
2. Selection of solid phase extraction column
Solid phase extraction is a sample preparation technology commonly used in the drug residue detection process, and is used for separating different components of a sample by filling solid chromatographic particles in a box-type device so as to achieve the purpose of enriching an object to be detected. For the purification of albendazole and its three metabolites, Waters Oasis MCX column and ProElutAL-A acidic Al were used for the comprehensive comparison in this experiment2O3The purification effects of three solid phase extraction columns, a Waters PRIME HLB column, were found when a Waters Oasis MCX column and acidic Al were used2O3During column purification, the recovery rate of albendazole and albendazole-2-aminosulfone is obviously reduced, and an interference peak exists, so that the purification effect is poor. The Waters PRIME HLB column can effectively remove interference of phospholipid and fatty pigment in a sample matrix, has good purification effect, does not interfere the peak emergence of a target object by an impurity peak, has high recovery rate, and can prolong the service life of the chromatographic column by the obtained pure extract. In addition, the test is equipped with no-water in the SPE extraction processThe oil vacuum pump and the anti-atomization glass vacuum tank control the extraction process by each independent valve, so that the extraction efficiency can be improved while the cross contamination is avoided.
3. Filter selection
In order to protect the chromatographic analysis system and prolong the service life of the chromatographic column, the test investigates the filtering effect of four needle filters, namely a PVDF hydrophobic needle filter, a hydrophilic PTFE needle filter, a water phase needle filter and an organic phase nylon needle filter after a target object is redissolved by a mobile phase. The results show that the results of the on-machine detection of the samples filtered by the water phase pin filter and the PVDF are not ideal, the signal loss is large, and the peak-shaped tailing is serious; the organic phase nylon needle type filter has larger aperture, can not effectively filter impurities, and compared with the hydrophilic PTFE needle type filter, the organic phase nylon needle type filter has better performance, not only provides clean extract, but also does not influence the peak shape of the object to be detected, has higher recovery rate, so the hydrophilic PTFE needle type filter (13mm multiplied by 0.22 mu m) is finally selected.
4. Optimization of chromatographic conditions
(1) Selection of stationary phase
The stationary phase (chromatographic column) is responsible for the separation and is the heart region of the liquid chromatography system. Generally, chromatographic columns can be divided into two main types, namely packed columns and open columns, and the packed columns are mainly used in the test process of liquid chromatography. Among reports on the detection of albendazole and the residues of three metabolites thereof in foods of animal origin, the most commonly used analytical columns are C18 columns and other types of chromatographic columns. In the test, an HSS T3 column, a CSH C18 column, a BEHC18 column and a Shield RP18 column are used for comparison, and after the comparison test, the results show that when the HSS T3 column and the CSH C18 column are used for analysis, no matter how the flow rate, the column temperature and other relevant parameters are adjusted, the target peaks of albendazole sulfoxide and albendazole-2-aminosulfone cannot reach the ideal separation degree, overlapping peaks are generated, and the purpose of separation detection cannot be achieved, so that the target peaks cannot be used as the analysis column of the test method. And a more severe solvent peak occurs during the experiment using the Shield RP18 column, thus causing the loss of the target peak of albendazole sulfoxide. Finally, after being optimized by other chromatographic conditions, mobile phase ratio and other parameters, ACQUITY is selected
Figure BDA0003226070510000101
The BEH C18 chromatographic column is an analytical column of the test, the peak shapes of the 4 target objects are sharp and symmetrical, the target objects are not interfered with the impurity peak of the sample tissue, the separation degree is good, the analysis time is within 6min, and the detection can be completed quickly.
In the test process, the protection of the chromatographic column is also an important link, so that the test cost can be reduced, unnecessary waste is avoided, and the test can be ensured to be carried out smoothly. The test uses a combination of an on-line filter plus a guard column to protect the chromatographic column and the entire chromatographic system. Impurities and particles of the sample are brought into the chromatographic column by the liquid phase system, so that the service life of the chromatographic column can be shortened, and if the impurities and the particles cannot be removed in the sample pretreatment process, the impurities and the particles are brought into the liquid phase system, on one hand, the automatic sample injection needle can be blocked during sample injection, and on the other hand, the service life of the chromatographic column can be greatly reduced when the impurities and the particles are brought into the chromatographic column by the mobile phase. Therefore, the test uses a stainless steel in-line filter with a 0.2 μm replaceable sieve plate installed in front of the guard column, which effectively intercepts the tiny particles that may clog the sieve plate of the column. In addition, the pressure is one of important parameters in a liquid phase system, and whether the on-line filter is blocked or not and the sieve plate can be replaced in time can be considered when the pressure is continuously increased in the test process. The use of guard column also can promote chromatographic column's life, though can reduce the column effect, prolong a bit analysis time, after taking into account economic benefits and analytical ability, this experiment final use with the same guard column of analytical column packing install additional before the analytical column, guarantee chromatographic system normal operating through the combination of on-line filter and guard column finally.
(2) Optimization of mobile phases
The mobile phase plays an important role in chromatographic systems, and therefore the mobile phase selection has a great influence on the separation and peak shape of the sample. Currently, in related researches on simultaneous detection of albendazole and three metabolites thereof in animal derived foods, an acetonitrile-formic acid water system, a methanol-formic acid water system and an acetonitrile-mixed phosphate system are used more frequently. In the test, methanol-formic acid water is tried to be used as a mobile phase, but the gradient elution is about 4min in the test process, so that large fluctuation occurs, and the chromatographic peak of albendazole is lost; the mobile phase of the acetonitrile-0.02 mol/L mixed phosphate system adopts gradient elution to separate a target object, the baseline drift is found to be serious, only the mobile phase is used for sample injection, and when the mobile phase passes through a detector, more impurity peaks exist, and the response value is also very high; in a test experiment of the standard substance, a target peak of albendazole-2-aminosulfone is covered by an impurity peak in a solvent, and ideal separation cannot be achieved by adjusting related parameters such as flow rate and the like. When the mobile phase contains phosphate, the wrong operation can cause the saline liquid to stay in the pump, the organic solvent volatilizes, and the crystal of the salt is separated out and then can damage parts such as a sealing ring, a one-way valve, a pump head and the like, so that abnormal pressure fluctuation and even blockage are caused. When the acetonitrile-formic acid water system is tried to be used as a mobile phase, all target objects can be detected without mutual interference, so the acetonitrile-formic acid water system is finally selected as the mobile phase. In addition, the elution methods of liquid chromatography mainly include isocratic elution and gradient elution. In comparison, the gradient elution has high requirements on the stability of the instrument, and time is required to balance the system between sample tasks to reach the initial mobile phase proportion, so that the phenomenon of limit drift is easy to occur, even ghost peaks are generated, and the test data are influenced. Isocratic elution can fully make up for the defects, and the whole separation process is stable. Because the polarities of the four components to be detected are not very different, an acetonitrile-0.2% formic acid aqueous solution system is adopted as a mobile phase in a follow-up test, and an isocratic elution mode is adopted to perform the test, so that the separation effects of different proportions of acetonitrile: 0.2% formic acid water (25: 75, 27: 73, 29: 71, 31: 69, 33: 67 and 35: 65) are investigated. The results show that: when the volume ratio of acetonitrile to 0.2% formic acid to water is 31: 69, the chromatogram base line is stable, no solvent peak exists, the separation degree of each target object is best, and the peak emergence time of albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone is stabilized at about 5.10, 2.10, 1.40 and 1.10 min. In the test, a phenomenon of tailing of a target peak is found, considering that albendazole and three metabolites thereof belong to weak alkaline substances, and a proper amount of triethylamine is added into the mobile phase to inhibit ionization, so that the phenomenon of tailing of a chromatographic peak is reduced, and the chromatographic peak with good symmetry is obtained. Therefore, in the experiment, the influence of adding different amounts (0.01%, 0.03%, 0.05%, 0.07% and 0.09%) of triethylamine to the mobile phase on the improvement of the chromatographic peak shape of the target object is investigated, and the effect of improving the peak tailing phenomenon by using 0.05% of triethylamine is found to be the best, and each target object obtains a chromatographic peak with good symmetry. Therefore, the final mobile phase condition of the test is that acetonitrile is mixed with 0.2% formic acid aqueous solution (containing 0.05% triethylamine) and the mixture is eluted at equal speed with 31: 69 (V/V).
(3) Optimization of fluorescence detector excitation and emission wavelengths
The fluorescence detector is a detector with strong selectivity and is only suitable for detecting substances with fluorescence properties, and the excitation wavelength and the emission wavelength are necessary parameters for fluorescence detection and directly influence the sensitivity and the selectivity of the detection. The test adopts fluorescence scanning of a microplate reader to respectively optimize the excitation wavelength and the emission wavelength of albendazole and three metabolites thereof, the optimal excitation wavelength and the optimal emission wavelength of albendazole and three metabolites thereof are obtained by scanning, the response values of albendazole and albendazole sulfoxide are low, so that the response values of all targets are comprehensively considered through on-machine test, and the test finally selects and detects the excitation wavelength and the emission wavelength which are 286nm and 335nm respectively.
(4) Optimisation of other conditions
The column oven is a temperature control instrument used for controlling the temperature of a chromatographic column to keep constant in the analysis of a liquid chromatograph, and generally, the column temperature is increased, the retention time of a target object is reduced, the resolution ratio is reduced, and the chromatographic separation process is accelerated. The test optimizes the temperature of the chromatographic column, and examines the peak appearance of the target object under the conditions of the column temperature (31 ℃, 33 ℃, 35 ℃ and 37 ℃). The results show that: when the column temperature is higher, the retention time of albendazole and albendazole sulfone is slightly advanced, but the chromatographic peaks of albendazole sulfoxide and albendazole-2-aminosulfone are crossed, so that the separation degree of the albendazole sulfoxide and the albendazole-2-aminosulfone is influenced; when the column temperature is 35 ℃, the separation degree of albendazole sulfoxide and albendazole-2-aminosulfone is highest and is not interfered with each other, all the target objects are completely detected within 6min, so the optimum column temperature is selected to be 35 ℃.
The column pressure of the column increases with increasing flow rate, and decreasing flow rate increases the column efficiency, but this increases the analysis time. The experiment inspects the influence of the flow rate on the peak-off time of a target peak, the retention time of albendazole is gradually advanced with the increase of the flow rate, but the amplitude is not large, and the improvement is not particularly obvious, while the separation degrees of albendazole sulfoxide and albendazole-2-aminosulfone are slightly reduced, but the retention time is not changed greatly. Therefore, the optimum flow rate is selected to be 0.25mL/min, taking into account the degree of separation between the target components and the time to peak.
(5) Comparison with Prior detection methods
Up to now, no ultra performance liquid chromatography-fluorescence detection method (UPLC-FLD) has been reported for simultaneous detection of albendazole and its three metabolite residues in pork, poultry and poultry eggs. The ultra-high performance liquid chromatography-fluorescence detection method (UPLC-FLD) established in the test can be used for simultaneously detecting the residues of albendazole and three metabolites thereof in pork, poultry and poultry eggs, the liquid-liquid extraction and solid-phase extraction combined technology is adopted for purifying the sample matrix, the recovery rates of albendazole and three metabolites thereof in all samples are more than 80.37%, the detection limit is 0.2-3.9 mu g/kg, the quantification limit is 1.0-12.6 mu g/kg, the detection sensitivity is improved, the detection process is completely completed within 6min, the working efficiency is greatly improved, the efficiency is high, the speed is high, new technical support is provided for the high-efficiency and quick detection of the residues of albendazole and three metabolites thereof in pork, poultry and poultry eggs, and the application value is high.
Table 2 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision (n ═ 6) in pork blank
Figure BDA0003226070510000141
Note: maximum residual limit of
Table 3 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision in chicken muscle blanks (n ═ 6)
Figure BDA0003226070510000151
Note: maximum residual limit of
Table 4 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision (n ═ 6) in duck muscle blanks
Figure BDA0003226070510000161
Note: maximum residual limit of
Table 5 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision in muscle of goose blanks (n ═ 6)
Figure BDA0003226070510000171
Note: maximum residual limit of
Table 6 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision (n ═ 6) in whole egg of blank chickens
Figure BDA0003226070510000181
Note: maximum residual limit of
Table 7 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision (n ═ 6) in duck egg white
Figure BDA0003226070510000191
Note: maximum residual limit of
Table 8 albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-aminosulfone addition recovery and precision (n ═ 6) in whole white goose eggs
Figure BDA0003226070510000201
Note: maximum residual limit of

Claims (10)

1. A method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs is characterized by comprising the following steps:
sequentially extracting pork, poultry meat and poultry eggs with an extractant, degreasing with n-hexane, purifying an extracting solution with an HLB solid phase extraction column, blowing the purified sample solution into a nitrogen blowing instrument with nitrogen for drying, and detecting by using a UPLC-FLD system after redissolution; the extracting agent adopted by the pork and the poultry meat is ethyl acetate, and the extracting agent adopted by the poultry eggs is formic acid-acetonitrile; the albendazole and three metabolites thereof are albendazole, albendazole sulfone, albendazole sulfoxide and albendazole-2-amino sulfone.
2. The method for simultaneously detecting albendazole and the multi-residue of three metabolites thereof in pork, poultry and poultry eggs according to claim 1, wherein the solid phase extraction process comprises: waters
Figure FDA0003226070500000011
Sequentially activating and balancing a PRIME HLB solid-phase extraction column by using methanol and ultrapure water; leaching the sample solution with a mobile phase, draining the extraction column, and eluting with ammoniated methanol; the ammoniated methanol is ammonia water, methanol, V/V, 2: 8.
3. The method for simultaneously detecting albendazole and the multi-residue of the three metabolites thereof in pork, poultry meat and poultry eggs as claimed in claim 1, wherein the pork, the poultry meat and the poultry eggs are sequentially extracted by an extracting agent, subjected to vortex oscillation and ultrasonic-assisted extraction for 5-10 min, centrifuged for 5-10 min, the supernatant is transferred after centrifugation, the extraction steps are repeated on the residues, and the two supernatants are combined and dried by nitrogen.
4. The method for simultaneously detecting albendazole and its three metabolite residues in pork, poultry and poultry eggs as claimed in claim 1, wherein ACQUITY is used
Figure FDA0003226070500000012
BEH C18The chromatographic column serves as an analytical column.
5. The method for simultaneously detecting albendazole and the multi-residue of three metabolites thereof in pork, poultry and eggs as claimed in claim 1, wherein the mobile phase A is acetonitrile; the mobile phase B is 0.2% formic acid water solution, and the mobile phase B contains 0.05% triethylamine.
6. The method for simultaneously detecting albendazole and the multi-residue of the three metabolites thereof in pork, poultry and poultry eggs as claimed in claim 1, wherein the fluorescence detector adopts a single-channel detection mode, and the wavelengths are set as follows: an excitation wavelength of 286nm and an emission wavelength of 335 nm.
7. The method for simultaneously detecting albendazole and the multi-residue of the three metabolites thereof in pork, poultry and eggs as claimed in claim 1, wherein the column temperature is 35 ℃ and the flow rate is 0.25 mL/min; the injection volume was 5. mu.L.
8. The method for simultaneously detecting albendazole and the multiple residues of the three metabolites thereof in pork, poultry and poultry eggs according to claim 1, wherein the elution mode is isocratic elution, and A: B: 31: 69 (V/V).
9. The method for simultaneously detecting albendazole and the multiple residues of the three metabolites thereof in pork, poultry and eggs as claimed in claim 1, wherein the extraction agent adopted by the eggs is 0.2% formic acid-acetonitrile.
10. The method for simultaneously detecting albendazole and the multi-residue of three metabolites thereof in pork, poultry meat and eggs as claimed in claim 1, wherein the detection formulas of pork, poultry meat and eggs are respectively:
Figure FDA0003226070500000021
CN202110972164.0A 2021-08-24 2021-08-24 Method for simultaneously detecting albendazole and multi-residue of three metabolites thereof in pork, poultry and poultry eggs Pending CN113702526A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108828082A (en) * 2018-04-18 2018-11-16 中南大学 A kind of detection method for extracting albendazole class compound from the flesh of fish

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108828082A (en) * 2018-04-18 2018-11-16 中南大学 A kind of detection method for extracting albendazole class compound from the flesh of fish

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
刁志祥: "猪肉、禽肉和禽蛋中阿苯达唑及其三种代谢产物残留同时检测的UPLC-FLD方法的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *

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