CN109655551A - It is a kind of efficiently detect birds, beasts and eggs simultaneously, Thiamphenicol, Florfenicol and the how remaining analysis method of florfenicol amine in poultry and pork - Google Patents
It is a kind of efficiently detect birds, beasts and eggs simultaneously, Thiamphenicol, Florfenicol and the how remaining analysis method of florfenicol amine in poultry and pork Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to field of veterinary drug residue detection, more particularly to it is a kind of efficiently detect birds, beasts and eggs simultaneously, Thiamphenicol, Florfenicol and the how remaining analysis method of florfenicol amine in poultry and pork, include the following steps: 1) birds, beasts and eggs, poultry or pork sample extraction and purification；2) ultra performance liquid chromatography-fluorescence detection: a. ultra performance liquid chromatography condition: with mobile phase A: the NaH containing 0.005mol/L2PO4, 0.003mol/L lauryl sodium sulfate and 0.05% triethylamine aqueous solution and Mobile phase B: acetonitrile is mobile phase；B. fluorescence detection.This method can efficiently, it is accurate detect simultaneously birds, beasts and eggs (egg, duck's egg, goose egg, pigeon eggs and quail egg), in poultry (chicken, duck and goose) and pork Thiamphenicol, Florfenicol and its metabolite florfenicol amine residual, and meet the requirement of the Ministry of Agriculture, the U.S. (FDA) and European Union (EU) detection of veterinary drugs in food method.
Description
Technical field
The present invention relates to field of veterinary drug residue detection, and in particular to a kind of efficiently to detect in birds, beasts and eggs, poultry and pork simultaneously
Thiamphenicol, Florfenicol and the how remaining analysis method of florfenicol amine.
Background technique
Currently, the detection method about Thiamphenicol, Florfenicol and its residues of metabolite florfenicol amine has both at home and abroad
Microbial method, immunological detection method, gas chromatography, high performance liquid chromatography, gas chromatography tandem mass spectrometry method and liquid phase color
Spectrum-tandem mass spectrometry etc., but ultra performance liquid chromatography-fluorescence detection (UPLC-FLD) detects birds, beasts and eggs, poultry and pork simultaneously
The method of middle Thiamphenicol, Florfenicol and its residues of metabolite florfenicol amine is not yet appeared in the newspapers.Therefore, the present invention is for the first time
Establish birds, beasts and eggs, Thiamphenicol, Florfenicol and its residues of metabolite florfenicol amine while the UPLC- of detection in poultry and pork
FLD detection method.
Summary of the invention
In order to it is efficient, accurate while detection Thiamphenicol, Florfenicol and its metabolite florfenicol amine be in birds, beasts and eggs
Residual in (egg, duck's egg, goose egg, pigeon eggs and quail egg), poultry (chicken, duck and goose) and pork, the present invention provide
A kind of ultra performance liquid chromatography-fluorescence detection (UPLC-FLD) method.This method can it is efficient, accurate while detection birds, beasts and eggs,
The residual of Thiamphenicol, Florfenicol and its metabolite florfenicol amine in poultry and pork, and meet the Ministry of Agriculture, European Union
(EU) requirement of detection of veterinary drugs in food.This method elution program is simple, and solvent consumption is few, and analysis efficiency is high, sensitivity and precision
It spends, is suitble to apply and popularization in batch samples analysis.
In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows: a kind of efficiently to detect birds, beasts and eggs, poultry simultaneously
And Thiamphenicol, Florfenicol and the how remaining analysis method of florfenicol amine in pork, which is characterized in that including walking as follows
It is rapid:
1) birds, beasts and eggs, poultry or pork sample extraction and purification；
2) ultra performance liquid chromatography-fluorescence detection:
A. ultra performance liquid chromatography condition: with ACQUITYBEH C18For chromatographic column；With mobile phase A: containing
The NaH of 0.005mol/L2PO4, 0.003mol/L lauryl sodium sulfate and 0.05% triethylamine aqueous solution and Mobile phase B:
Acetonitrile is mobile phase；Flow velocity: 0.2mL/min；Column temperature: 30 DEG C；The percentage is percent by volume；
B. fluorescence detector, 233 ± 2nm of excitation wavelength, 284 ± 2nm of launch wavelength fluorescence detection: are used.
In step a, the pH value of mobile phase A is 5.3 ± 0.1.In step a, mobile phase A when separating object in birds, beasts and eggs: B
=64:36V/V, mobile phase A when separating object in poultry and pork: B=65:35V/V；The object nail sulfone is mould
Element, Florfenicol or florfenicol amine.In step a, sampling volume preferably 10 μ L.
Step (1) detailed process are as follows: 1) accurately weigh the good birds, beasts and eggs of homogeneous, poultry or pork blank sample and be put in mortar
In, and appropriate diatomite grinding is added, it is fitted into abstraction pool, is extracted with ASE；2) in 1500psi, 80 DEG C of extraction conditions
Under, birds, beasts and eggs sample acetonitrile: ammonium hydroxide extraction, acetonitrile: the volume ratio of ammonium hydroxide is 98:2, poultry and pork sample ethyl acetate:
Ammonium hydroxide extraction, ethyl acetate: the volume ratio of ammonium hydroxide is 98:2, and static extracting 2 times, each static extracting time 3min, nitrogen is blown
60s is swept, it is stand-by to collect extract liquor；3) above-mentioned extract liquor is transferred to centrifuge tube, is placed in 50 DEG C of centrifuge concentrators and is concentrated；Extraction
After liquid concentration, pure acetonitrile is added, vortex oscillation mixes, and n-hexane whirlpool is added and mixes, and degreasing is stood, and discards upper layer, and repetition is gone
Rouge is primary, is then placed in centrifuge concentrator and is concentrated to dryness again, for use；4) sample of the concentration after dry is answered with 35% acetonitrile
Molten, vortex oscillation mixes, and is centrifuged, and filtering, filtrate is for ultra performance liquid chromatography-fluorescence detection.
The present invention compares the acetonitrile of different volumes ratio: ammonium hydroxide (99:1,98:2,97:3,96:4,95:5, V/V) is fowl
In egg when extractant, acetonitrile: ammonium hydroxide (98:2, V/V), ethyl acetate: ammonium hydroxide (98:2, V/V) and acetonitrile: ethyl acetate: ammonium hydroxide
Method rate of recovery when (49:49:2, V/V) is extractant in poultry and pork, the results showed that with acetonitrile in birds, beasts and eggs: ammonium hydroxide
Ratio is 98:2, when V/V, with ethyl acetate in poultry and pork: when ammonium hydroxide (98:2, V/V) is used as extractant, target compound
Rate of recovery highest.This test also provides a comparison of influence of the different ASE Extracting temperatures to extraction recovery, and discovery temperature is 80 DEG C
When extraction recovery highest, extraction effect is best.Therefore, final choice Extracting temperature is 80 DEG C, with acetonitrile: ammonium hydroxide (98:2,
It V/V is) extractant in birds, beasts and eggs, ethyl acetate: ammonium hydroxide (98:2, V/V) is the extractant in poultry and pork, the extracting method sample
The influence of product matrix is small, extraction efficiency is high, the rate of recovery is high.
Compared with the existing technology, the invention has the benefit that
ACQUITY is used in the present inventionBEH C18(2.1 × 50mm, 1.7 μm) chromatographic column, (is contained with water
The NaH of 0.005mol/L2PO4Solution, 0.003mol/L lauryl sodium sulfate and 0.05% triethylamine, with 85% H3PO4
Adjust pH to 5.3 ± 0.1)-acetonitrile system be mobile phase (birds, beasts and eggs: 64:36, V/V；Poultry and pork: 65:35, V/V), flow velocity is
0.2mL/min, chromatographic peak peak type is sharp, and analyte retention time is moderate, no other impurity peaks interference.
The present invention provides a kind of ultra performance liquid chromatography-fluorescence detection (UPLC-FLD) methods.The present invention is imitated in analysis
Rate, solvent consumption, the rate of recovery, precision, sensitivity and reproducibility angle, are compared with other detection methods, discovery we
Method analysis efficiency is higher, mobile phase consumption less, the rate of recovery, accuracy and high sensitivity, favorable reproducibility, be suitble in batch sample point
It is applied and popularization in analysis.This method efficient, accurate can detect simultaneously birds, beasts and eggs (egg, duck's egg, goose egg, pigeon eggs and quail egg), fowl
The residual of Thiamphenicol, Florfenicol and its metabolite florfenicol amine in meat (chicken, duck and goose) and pork, and meet
The requirement of the Ministry of Agriculture, the U.S. (FDA) and European Union (EU) detection of veterinary drugs in food method.
Detailed description of the invention
Fig. 1 standard items, blank egg and blank egg sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 2 standard items, blank duck's egg and blank duck's egg sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 3 standard items, blank goose egg and blank goose egg sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 4 standard items, blank pigeon eggs and blank pigeon eggs sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 5 standard items, blank quail egg and blank quail egg sample add standard items (Thiamphenicol: 25 μ g/kg, fluorobenzene
Buddhist nun examines and florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 6 standard items, blank chicken and blank chicken meat sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 7 standard items, blank duck and blank duck sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram
Fig. 8 standard items, blank goose and blank goose sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
Fig. 9 standard items, blank pork and blank pork sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol
And florfenicol amine: 50 μ g/kg) chromatogram；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 10 egg；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 11 duck's egg；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 12 goose egg；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 13 pigeon eggs；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 14 quail egg；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 15 chicken；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 16 duck；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 17 goose；
The matrix of Thiamphenicol, Florfenicol and florfenicol amine adds standard curve in Figure 18 pork.
Specific embodiment
Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified
The meaning of understanding.
Combined with specific embodiments below, and referring to data the present invention is described in further detail.These embodiments only be
It illustrates the present invention, rather than limits the scope of the invention in any way.
Below in an example, the various processes and method being not described in detail are conventional methods as known in the art.
The source of agents useful for same, trade name and it is necessary to list its constituent person, are indicated on the first occurrence, thereafter phase used
Unless otherwise specified with reagent, it is the same as indicated for the first time.
1. testing the raising and sample acquisition of livestock and poultry
The lay eggs yellow chicken 30 (Jiangsu Jinghai Poultry Group Co., Ltd.) in capital sea, 28-30 week old of 28 week old is chosen respectively to produce
Egg Gaoyou duck 30 (Jiangsu Gaoyou Duck Group), 30 week old lay eggs Yangzhou goose 48 (Yangzhou Tian Gee industry Development Co., Ltd),
35 week old lay eggs pigeon 50 (Jiangsu Jiangnan Ge Ye Co., Ltd), 20 week old lay eggs quail 30 (Xinghua City pacify Fengzhen quail
Cultivate Co., Ltd), pre- before testing to raise 1 week, feeding is free of complete feed (the limited public affairs of Jiangsu Jing Hai Qin Ye group of any drug
Department's feed factory and Yangzhou are raised big feed factory and are provided), free water.The full price for being free of any antibacterials is fed during test
Feed collects birds, beasts and eggs in daily 17:30-18:00, and continuous to collect 10 days, birds, beasts and eggs sample is stored in low temperature egg library.Sample collection
It will be dispensed after egg, duck's egg, goose egg, pigeon eggs and quail egg sample homogenization respectively afterwards, post label, respectively as blank sample,
It is placed in -34 DEG C of refrigerators and saves.
Randomly select the yellow chicken (Jiangsu Jinghai Poultry Group Co., Ltd.) in 112 age in days capital sea, 142 age in days Gaoyou duck (Jiangsu
Gaoyou duck group), 70 age in days Yangzhou geese (Jiangsu Tian Gee industry Development Co., Ltd) each 6 (male and female is fifty-fifty) and 180 ages in days
Ternary (Du × length × big) hybridized pig (Changzhou health and happiness Farming Ltd.) 6 (male and female is fifty-fifty), single cage (column) raising, feeding
Complete feed (Jiangsu Jinghai Poultry Group Co., Ltd.'s feed factory and Yangzhou are raised big feed factory and provided) without any drug,
Free water.It is butchered after raising 2 weeks, takes the chest muscle meat for examination chicken, duck, goose respectively, the hind leg muscle of pig blends it simultaneously respectively
It mixes well as blank sample, dispenses, sealing is placed in -34 DEG C of refrigerators and saves backup.
2. the present invention extracts and purifying step
1) the good birds, beasts and eggs of (2.0 ± 0.02) g homogeneous, poultry or pork blank sample are accurately weighed to be put in mortar, and is added
Enter appropriate diatomite grinding, in order to reach optimal extraction efficiency, sample is ground into little particle as far as possible, is packed into the extraction of 22mL
It takes in pond, is extracted with ASE；
2) in 1500psi, 80 DEG C (test compares 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C and 120 DEG C, optimum temperature 80
DEG C, temperature range equally meets recovery of extraction verifying in 80 ± 2 DEG C and requires) under extraction conditions, birds, beasts and eggs sample acetonitrile: ammonia
Water (98:2, V/V) extraction, poultry and pork sample ethyl acetate: ammonium hydroxide (98:2, V/V) extraction, static extracting 2 times, every time
Static extracting time 3min (best static extracting 2 times and best static extracting time 3min, it can be ensured that all objects are fine
Extract on ground), nitrogen purges 60s, and it is stand-by to collect extract liquor；
3) above-mentioned extract liquor is transferred to 50mL centrifuge tube, is placed in 50 DEG C of centrifuge concentrators (2000 × g) and is concentrated into 1-
2mL；After extract liquor concentration, the pure acetonitrile of 1mL is added, vortex oscillation mixes 1min, and 10mL n-hexane (being saturated with pure acetonitrile) is added
Whirlpool mixes, degreasing, stands 5min, discards upper layer, and repetition degreasing is primary, is then placed in again in centrifuge concentrator (2000 × g)
It is concentrated to dryness, for use；
4) the sample acetonitrile of 2.0mL 35% by concentration after dry redissolves, and vortex oscillation mixes, 12100 × g centrifugation
15min, crosses 0.22 μm of PVDF pin type filter, and filtrate is detected for UPLC-FLD.
3. ultra performance liquid chromatography condition
Chromatographic column: ACQUITYBEH C18, 2.1mm × 100mm, 1.7 μm；Mobile phase A: ultrapure water (contains
The NaH of 0.005mol/L2PO4Solution, 0.003mol/L lauryl sodium sulfate and 0.05% triethylamine, with 85% H3PO4
Adjust pH to 5.3 ± 0.1), Mobile phase B: acetonitrile；The isocratic elution program that this research uses, A:B=when separating object in birds, beasts and eggs
64:36 (V/V), A:B=65:35 (V/V) when separating object in poultry and pork；Detector: fluorescence detector, optimization aim
The excitation wavelength and launch wavelength of object, determine best detection wavelength: excitation wavelength 233nm, launch wavelength 284nm, in this wavelength
Under not only ensure that the high response of object, but also guarantee free from admixture interference (233 ± 2nm of excitation wavelength, launch wavelength 284 ±
2nm is able to satisfy testing requirements)；Flow velocity: 0.2mL/min；Sampling volume: 10 μ L；Column temperature: 30 DEG C.
4 quantitative approach
The drafting of 4.1 standard curves
Egg, duck's egg, goose egg, quail egg, pigeon egg, chicken that 10 parts of (2.0 ± 0.02) g have been homogenized are accurately weighed respectively
Meat, duck, goose and pork blank sample are extracted by 2. present invention and are carried out with the method in purifying step to above-mentioned blank sample
It extracts and purifies, prepare blank sample matrix extracting solution, it is spare.
Appropriate Thiamphenicol, Florfenicol and florfenicol amine standard working solution is taken to be added to bare substrate extracting solution respectively
In, so that the final corresponding methyl sulfone in blank egg, duck's egg, goose egg, quail egg, pigeon egg, chicken, duck, goose and pork
The addition concentration of mycin is the μ g/kg of LOQ, 20.0,50.0,100.0,150.0,200.0,250.0, the addition concentration of Florfenicol
For the μ g/kg of LOQ, 20.0,50.0,100.0,200.0,300.0,400.0, the addition concentration of florfenicol amine be LOQ, 10.0,
20.0、50.0、100.0、200.0、400.0μg/kg。
Each concentration samples of above-mentioned processing are analyzed under the conditions of the UPLC-FLD optimized.It is mould with methyl sulfone respectively
It is independent variable (x) that element, Florfenicol and florfenicol amine standard working solution add concentration in different bare substrates, and peak area is
Dependent variable (y) draws standard curve.
By table 1 and attached drawing 10-18 as it can be seen that Thiamphenicol, Florfenicol and florfenicol amine are in blank birds, beasts and eggs, poultry and pig
Concentration is added in meat sample product respectively within the scope of LOQ-250.0 μ g/kg, LOQ-400.0 μ g/kg and LOQ-400.0 μ g/kg,
Peak area (y-axis) and concentration (x-axis) are in good linear relationship；The range of linearity, equation of linear regression and coefficient of determination R2See
Table 1.It, need to be by the concentration dilution of analysis to the knot within the scope of this, detected if the concentration of analysis is more than the range of linearity of sample
Fruit obtains the concentration of raw sample multiplied by extension rate.
Thiamphenicol, the range of linearity of Florfenicol and florfenicol amine, regression equation in 1 birds, beasts and eggs of table, poultry and pork
And the coefficient of determination
The measurement of 4.2 rate of recovery and precision
The homogeneous good blank sample of (2.0 ± 0.02) g is accurately weighed, produces bare substrate extracting solution by the method in 2.Point
It does not take appropriate Thiamphenicol, Florfenicol and florfenicol amine standard working solution to be added in bare substrate extracting solution, makes it most
It is LOQ, 0.5MRL, 1.0MRL, 2.0MRL that concentration is added in each blank sample eventually, is extracted by the method in 2, Mei Getian
Add concentration to set 6 parallel, then detected for UPLC-FLD, finally will test result and bring into and acquire concentration in standard curve, with reality
The concentration of the analyte of addition is compared and acquires TIANZHU XINGNAO Capsul.
Standard items, blank egg and blank egg sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol and fluorine
Benzene Buddhist nun examines amine: 50 μ g/kg) chromatogram is shown in Fig. 1；(methyl sulfone is mould for standard items, blank duck's egg and blank duck's egg sample addition standard items
Element: 25 μ g/kg, Florfenicol and florfenicol amine: 50 μ g/kg) chromatogram is shown in Fig. 2；Standard items, blank goose egg and blank goose egg
Sample addition standard items (Thiamphenicol: 25 μ g/kg, Florfenicol and florfenicol amine: 50 μ g/kg) chromatogram is shown in Fig. 3；Standard
Product, blank pigeon eggs and blank pigeon eggs sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol and florfenicol amine: 50 μ
G/kg) chromatogram is shown in Fig. 4；Standard items, blank quail egg and blank quail egg sample add standard items (Thiamphenicol: 25 μ g/
Kg, Florfenicol and florfenicol amine: 50 μ g/kg) chromatogram is shown in Fig. 5；Standard items, blank chicken and the addition of blank chicken meat sample
Standard items (Thiamphenicol: 25 μ g/kg, Florfenicol and florfenicol amine: 50 μ g/kg) chromatogram is shown in Fig. 6；Standard items, blank
Duck and blank duck sample add standard items (Thiamphenicol: 25 μ g/kg, Florfenicol and florfenicol amine: 50 μ g/kg) color
Spectrogram is shown in Fig. 7；Standard items, blank goose and blank goose sample addition standard items (Thiamphenicol: 25 μ g/kg, Florfenicol and
Florfenicol amine: 50 μ g/kg) chromatogram is shown in Fig. 8；(methyl sulfone is mould for standard items, blank pork and blank pork sample addition standard items
Element: 25 μ g/kg, Florfenicol and florfenicol amine: 50 μ g/kg) chromatogram is shown in Fig. 9.
Withinday precision: above-mentioned 4 are added in same instrument of different time points on the same day and same standard curve
Concentration is measured, and each addition concentration sets 6 in parallel, finds out in a few days RSD.
Day to day precision: in 7 days not on the same day, various criterion curve (daily all draw standard curve) same instrument pair
Above-mentioned 4 additions concentration is measured, and each addition concentration sets 6 in parallel, finds out RSD in the daytime.
With this condition, Thiamphenicol, Florfenicol and Florfenicol in the method for the present invention extraction birds, beasts and eggs, poultry and pork
The TIANZHU XINGNAO Capsul and precision of amine are shown in Table 2- table 10.
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 2 blank egg of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 3 blank duck's egg of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 4 blank goose egg of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 5 blank pigeon eggs of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 6 blank quail egg of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 7 blank chicken muscle of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 8 blank duck muscle of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 9 blank goose muscle of table
Note: α maximum residue limit
Note: α .Maximum Residue Limits
Thiamphenicol, Florfenicol and florfenicol amine TIANZHU XINGNAO Capsul and precision (n=6) in 10 blank pork of table
Note: α maximum residue limit
The determination of Note: α .Maximum Residue Limits4.3 detection limit and quantitative limit
The homogeneous good blank sample of (2.0 ± 0.02) g is accurately weighed, produces bare substrate extracting solution by the method in 2.With
Bare substrate extracting solution dilutes the Thiamphenicol, Florfenicol and florfenicol amine standard working solution of low concentration step by step, carries out
UPLC-FLD detection, repeats sample introduction 6 times, calculates signal-to-noise ratio (S/N).As S/N >=3, corresponding target compound concentration is to examine
Limit (LOD) is surveyed, corresponding target compound concentration is quantitative limit (LOQ) as S/N >=10.
According to the addition recovery test of above-mentioned 6 parallel blank samples, obtain under existence conditions, TAP is in birds, beasts and eggs, poultry
With the LOD in pork be respectively 3.3,3.4,3.4,3.4,3.3,3.0,3.3,3.1,3.5 μ g/kg, LOQ be respectively 9.7,9.9,
9.8,9.9,9.7,8.5,9.0,8.8,9.4μg/kg.LOD of the FF in birds, beasts and eggs, poultry and pork be respectively 4.7,4.9,4.8,
4.8,4.7,3.6,4.2,3.8,3.3 μ g/kg, LOQ be respectively 10.5,11.7,11.2,11.2,10.6,10.2,11.4,
10.6,9.8μg/kg.LOD of the FFA in birds, beasts and eggs, poultry and pork be respectively 1.8,1.9,1.9,1.9,1.8,1.4,1.3,
1.0,1.2 μ g/kg, LOQ are respectively 4.3,4.7,4.7,4.8,4.6,3.5,3.3,3.0,3.2 μ g/kg.Reality of the present invention
Example is the description of the invention and cannot limit the present invention, with the comparable meaning and scope of the present invention in any change and tune
It is whole, it is all considered as within the scope of the invention.
Claims (6)
- Efficiently detect that birds, beasts and eggs, how remaining Thiamphenicol, Florfenicol and florfenicol amine be in poultry and pork simultaneously 1. a kind of Analysis method, which comprises the steps of:1) birds, beasts and eggs, poultry or pork sample extraction and purification；2) ultra performance liquid chromatography-fluorescence detection:A. ultra performance liquid chromatography condition: with ACQUITY UPLC® BEH C18For chromatographic column；With mobile phase A: containing 0.005 The NaH of mol/L2PO4, 0.003 mol/L lauryl sodium sulfate and 0.05% triethylamine aqueous solution and Mobile phase B: acetonitrile For mobile phase；Flow velocity: 0.2 mL/min；Column temperature: 30 °C；B. fluorescence detector, excitation wavelength 233 ± 2 nm, 284 ± 2nm of launch wavelength fluorescence detection: are used.
- 2. it is according to claim 1 it is a kind of efficiently detect birds, beasts and eggs simultaneously, Thiamphenicol, Florfenicol in poultry and pork With the how remaining analysis method of florfenicol amine, which is characterized in that in step a, the pH value of mobile phase A is 5.3 ± 0.1.
- 3. it is according to claim 1 it is a kind of efficiently detect birds, beasts and eggs simultaneously, Thiamphenicol, Florfenicol in poultry and pork With the how remaining analysis method of florfenicol amine, which is characterized in that in step a, mobile phase A when separating object in birds, beasts and eggs: B =64:36 V/V, mobile phase A when separating object in poultry and pork: B=65:35 V/V；The object nail sulfone is mould Element, Florfenicol or florfenicol amine.
- 4. it is according to claim 1 it is a kind of efficiently detect simultaneously birds, beasts and eggs, Thiamphenicol in poultry and pork, Florfenicol and The how remaining analysis method of florfenicol amine, which is characterized in that step (1) detailed process are as follows: 1) accurately weigh the good fowl of homogeneous Egg, poultry or pork blank sample are put in mortar, and appropriate diatomite grinding is added, and are fitted into abstraction pool, are extracted with ASE It takes；2) at 1500 psi, 80 °C of extraction conditions, birds, beasts and eggs sample acetonitrile: ammonium hydroxide extracts, acetonitrile: the volume ratio of ammonium hydroxide is 98:2, poultry and pork sample ethyl acetate: ammonium hydroxide extraction, ethyl acetate: the volume ratio of ammonium hydroxide is 98:2, static extracting 2 Secondary, each 3 min of static extracting time, nitrogen purges 60 s, and it is stand-by to collect extract liquor；3) above-mentioned extract liquor is transferred to centrifugation Pipe, is placed in 50 °C of centrifuge concentrators and is concentrated；After extract liquor concentration, pure acetonitrile is added, vortex oscillation mixes, and n-hexane is added Whirlpool mixes, degreasing, stands, and discards upper layer, and repetition degreasing is primary, is then placed in centrifuge concentrator and is concentrated to dryness again, for use； 4) 35% acetonitrile of sample by concentration after dry redissolves, and vortex oscillation mixes, and is centrifuged, and filtering, filtrate is for ultra high efficiency liquid phase color Spectrum-fluorescence detection.
- 5. it is according to claim 1 it is a kind of efficiently detect simultaneously birds, beasts and eggs, Thiamphenicol in poultry and pork, Florfenicol and The how remaining analysis method of florfenicol amine, which is characterized in that in step a, the percentage is percent by volume.
- 6. it is according to claim 1 it is a kind of efficiently detect birds, beasts and eggs simultaneously, Thiamphenicol, Florfenicol in poultry and pork With the how remaining analysis method of florfenicol amine, which is characterized in that in step a, sampling volume: 10 μ L.
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