CN106770789A - Detect simultaneously broiler chicken liver, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography method - Google Patents
Detect simultaneously broiler chicken liver, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses it is a kind of and meanwhile detect broiler chicken liver, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography method, belong to the Ultra Performance Liquid Chromatography detection field of aflatoxin content.The present invention detect simultaneously broiler chicken liver, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography method, comprise the following steps：(1) AFB1 and M1 in liver, kidney or the Chicken Tissues of broiler chicken to be detected are extracted, extract solution is obtained；(2) extract solution purified, derived；(3) qualitative, quantitative measure is carried out using ultra-performance liquid chromatography.The inventive method makes AFB1 and M1 in broiler chicken liver, kidney or chicken obtain fully extracting separation, have the advantages that the good degree of accuracy, reappearance stabilization, sensitivity are high, quick, can be applied to detect the aflatoxin residual in animal edible tissues.
Description
Technical field
The inspection of AFB1 and Aflatoxins M1 content the present invention relates to broiler chicken liver, kidney and in Chicken Tissues
Survey method, more particularly to simultaneously detection broiler chicken liver, kidney and in Chicken Tissues AFB1 and M1 contents ultra high efficiency
Liquid-phase chromatography method, belongs to the Ultra Performance Liquid Chromatography detection field of aflatoxin content.
Background technology
Aflatoxin (Aflatoxins, abbreviation AFT), is that a group produced by fungi aspergillus flavus and aspergillus parasiticus is malicious by force
Property secondary metabolite, including AFB1, B2, G1, G2, especially AFB1 be strong polluter, its
Have a very wide distribution, in mainly producing and being present in the foodstuff grain or its article of manufacture of moldy metamorphism.Animal edible contains Huang
After the feed of aspertoxin B1 (AFB1), Aflatoxins M1 (AFM1) can be produced through hydroxylation metabolism in vivo, the two has
There is extremely strong toxicity, the mankind particularly children, the poisoning of various letting animals feeds can be caused, moreover it is possible to carcinogenic, teratogenesis, mutagenesis, i.e.,
The amount containing tens micrograms still has very big toxicity in making every kilogram of material, and the two can residue in liver, kidney, muscle of animal etc.
Mankind's edible portion, the health to the mankind, poultry, domestic animal causes greatly threat.
Ultra Performance Liquid Chromatography method (UPLC) is a brand-new classification in separation science, and it is by high performance liquid chromatography
The theory and principle of method (HPLC), cover little particle filler, the brand new technical such as very low system bulk and quick detection means,
Increased the flux of analysis, sensitivity and chromatogram peak capacity.Compared with traditional HPLC, the sensitivity of UPLC and separating degree are carried
Several times even decades of times high, and analysis time is shortened, solvent load is reduced, analysis cost is reduced, it is a kind of ratio
HPLC more rapidly, more efficient, more sensitive method for detecting residue.Therefore, a kind of accurate, sensitive, efficient ultra high efficiency liquid is set up
Aflatoxin residual in phase chromatography detection broiler chicken edible tissues, to ensureing the safety of animal products and safeguarding the mankind's
Health is respectively provided with important meaning.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of liver of detection broiler chicken simultaneously, kidney and aspergillus flavus in chicken
The Ultra Performance Liquid Chromatography method of toxin B1 (AFB1) and Aflatoxins M1 (AFM1) content, the method obtains AFB1 and AFM1
Separated to sufficiently extracting, have the advantages that the good degree of accuracy, reappearance stabilization, sensitivity are high.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention disclose first it is a kind of and meanwhile detect broiler chicken liver, kidney and in chicken AFB1 and AFM1 contents superelevation
Effect liquid phase chromatogram method, comprises the following steps：(1) AFB1 in liver, kidney and/or the Chicken Tissues of broiler chicken to be detected is extracted
And AFM1, obtain extract solution；(2) extract solution purified, derived；(3) qualitative, quantitative is carried out using Ultra Performance Liquid Chromatography
Determine.
AFB1 and AFM1 in step (1) liver for extracting broiler chicken to be detected, kidney and/or Chicken Tissues include：
By the liver of broiler chicken to be detected, kidney and/or Chicken Tissues crush, add Extraction solvent, it is sonicated after, shaking table concussion,
Anhydrous sodium sulfate is subsequently adding, is centrifuged, take supernatant.Wherein, the Extraction solvent is molten selected from dichloromethane, 70%-100% methyl alcohol
Any one in liquid or 84% acetonitrile solution (percent by volume), preferably dichloromethane；It is further preferred that according to g/ml
Meter, the liver of the broiler chicken to be detected, kidney or Chicken Tissues：Extraction solvent=1：10.The time of the ultrasound is 10min；
The power of ultrasound is 60Hz；The time of the shaking table concussion is 30-90min, preferably 60min；The rotating speed of the shaking table concussion
It is 400r/min.According to mass ratio meter, the liver of the broiler chicken to be detected, kidney and/or Chicken Tissues：Anhydrous sodium sulfate=1：
1。
Step (2) purification includes：By extract solution by Aspergillus flavus toxin immuno-affinity column, eluent is collected；Wherein
The Aspergillus flavus toxin immuno-affinity column is selected from any one in AFM1 immune affinity columns or AFB1 immune affinity columns, preferably
AFM1 immune affinity columns.Specific purifying step includes：Extract solution 10ml is taken, nitrogen drying under 50 DEG C of water bath conditions adds 2ml
Methyl alcohol dissolved residue, adds pH7.4 PBS solution 13mL, by AFM1 immune affinity columns, 1-2 drops/sec of flow velocity, the punching of 10ml pure water
Immune affinity column is washed, residual moisture in press-in air discharge post adds 1ml methyl alcohol wash-out, collects whole eluents.
Step (2) the derivative method is that (referring to document, " high performance liquid chromatography is to meat for trifluoroacetic acid pre-column derivatization
The measure of AFB1 residual quantity in chicken tissues sample ",《HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE》Science and technology version, in October, 2015 (on)
281-287.)；Product after derivative is dried up through nitrogen, constant volume, for loading.Specifically include：Eluent is dried up through nitrogen,
Trifluoroacetic acid, n-hexane are added, water-bath derives, then nitrogen drying, constant volume；Further eluted with 1ml methyl alcohol specifically, collecting
Whole eluents, nitrogen drying under 50 DEG C of water bath conditions adds 100 μ l trifluoroacetic acids, 200 μ l n-hexanes, and 40 DEG C of water-baths spread out
Raw 15min, nitrogen drying under 50 DEG C of water bath conditions.The solution of the constant volume is 10%-100% methanol aqueous solutions or 10%-
100% acetonitrile solution, preferably 10% methanol aqueous solution (percent by volume).
The condition of step (3) described Ultra Performance Liquid Chromatography includes：Chromatographic column is Waters Acquity UPLC BEH
C18, specification is 50mm × 2.1mm × 1.7 μm；Flow velocity 0.1-0.3mL/min；Sample size is 10 μ L；Mobile phase is acetonitrile：Water；
20-30 DEG C of column temperature；Excitation wavelength 365nm, launch wavelength 435nm；Run time 4min.According to volume basis, the mobile phase
It is acetonitrile：Water=15-30：The ratio of 70-85, i.e. acetonitrile and water is 15：85、20：80、25：75 or 30：70；Preferably, it is described
Mobile phase is acetonitrile：Water=20：80；The column temperature is preferably 25 DEG C；The flow velocity is preferably 0.2mL/min.
Step (3) qualitative, quantitative is determined to be included：According to the reservation of aflatoxin to be detected and aflatoxin standard items
Time consistency, is defined as target aflatoxin.Linear regression is done according to aflatoxin standard concentration and chromatographic peak area,
Obtain equation of linear regression；By the liver of broiler chicken to be detected, kidney or the peak area of aflatoxin brings phase into Chicken Tissues
The equation of linear regression answered, calculates the respective concentration of target aflatoxin.
Abundant extraction point of the extraction organic solvent for the AFB1 in the liver of broiler chicken, kidney or Chicken Tissues and AFM1
From with material impact.According to the property of aflatoxin, general extraction organic solvent mainly has methyl alcohol, dichloromethane and second
Nitrile.The present invention is compared to three kinds of organic solvents to the extraction effect of aflatoxin, wherein 80% methyl alcohol and 84% second
Nitrile is the common solvent for extracting aflatoxin in feed, but for extracting aspergillus flavus poison during liver, kidney or muscle etc. are organized
The rate of recovery of element is substantially less than dichloromethane.And, the recovery of standard addition of AFB1 and AFM1 shows when dichloromethane is Extraction solvent
Write the methanol solution higher than various concentrations.Therefore, present invention selection dichloromethane is the Extraction solvent of AFB1 and AFM1 in tissue.
Concussion extraction time there is also on recovery of standard addition and significantly affect.In the range of 30-90min, when extraction is shaken
Between for 60min when, the recovery of standard addition pole of AFB1 and AFM1 is significantly higher than concussion and extracts 30-50min；When continuing to extend concussion
Between to 70-90min, AFB1 and AFM1 recovery of standard addition with concussion 60min differences it is not notable.Therefore, the preferred concussion of the present invention
Extraction time is 60min.
The present invention to purification extract solution with immune affinity column investigated.The immune affinity column pair of Aflatoxins M1
The rate of recovery of AFM1 and AFB1 is very high, and AFB1 immune affinity columns can not adsorb AFM1.Therefore present invention selection AFM1 is immunized
Affinity column.The optimum results of constant volume solution show that organic phase selects methyl alcohol and ratio is at 10%, and chromatographic peak peak type is good, molten
Agent peak disturbs small to AFM1 peak types, and peak area is big.
The present invention is optimized to chromatographic condition including mobile phase, flow velocity, column temperature and Detection wavelength.Optimization of mobile phase
Result shows, mobile phase acetonitrile：Water is 20：AFB1 and AFM1 peak areas are noticeably greater than other ratio mobile phases under the conditions of 80.Stream
Fast optimum results show that AFB1 and AFM1 peak areas are noticeably greater than flow velocity for 0.1 or 0.3ml/min when flow velocity is 0.2ml/min
(P<0.01), and peak type is good, chromatogram peak energy is preferably separated；And flow velocity appearance time evening, and peak type when being 0.1ml/min
It is low flat；When flow velocity is 0.3ml/min, appearance time too early, is separated not thorough with solvent peak.Column temperature optimum results show, in post
Good separating effect when temperature is 25 DEG C, peak type is good, and peak area is big, with other treatment comparing differences significantly (P<0.01).The present invention is comprehensive
AFB1, AFM1 maximum absorption wavelength are closed, 435nm is used as final launch wavelength for selection, and 365nm is used as final excitation wavelength.
The inventive method is suitable for detecting the AFB1 and AFM1 of the liver of broiler chicken, kidney or muscle any one or more
Content.
Using the inventive method detection broiler chicken liver, kidney and in muscle, the average recovery rate of AFB1 is respectively
82.32%~85.56%, 85.34%~88.45% and 84.88%~89.73%；Precision respectively 1.47%~
4.45%th, 1.12%~3.54% and 2.01%~4.17%.Broiler chicken liver, kidney and in muscle Aflatoxins M1 it is average
The rate of recovery is respectively 92.17%~95.03%, 94.12%~97.21% and 95.32%~98.54%；Precision is respectively
1.83%~3.46%, 1.47%~3.81% and 1.96%~3.97%.AFB1 detection is limited to 0.008ng/mL,
Quantitatively it is limited to 0.02ng/mL；Aflatoxins M1 detection is limited to 0.003ng/mL, is quantitatively limited to 0.01ng/mL.
Detected simultaneously using the present invention broiler chicken liver, kidney and in chicken AFB1 and M1 contents ultra high efficiency liquid
Phase chromatographic process, detected in the liver, kidney and Chicken Tissues in AFB1 sub-chronic intoxication broiler chicken AFB1 and
The residual of AFM1.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention detect simultaneously broiler chicken liver, kidney and in chicken AFB1 and M1 contents ultra high efficiency liquid phase color
Spectral method, realizes that the abundant extraction of AFB1 and AFM1 is separated, with the degree of accuracy is good, reappearance stabilization, sensitivity it is high, quick etc. excellent
Point.Compared with the method for detecting residue of aflatoxin in current feed or grain, the sensitivity of the inventive method is high, the rate of recovery
Height, more easily and fast.The present invention for broiler chicken liver, kidney and in muscle AFB1 and AFM1 qualitative, quantitative and quick detection
There is provided easy, sensitive, the accurate method of one kind, in aflatoxin metabolism research, residual inspection and Safety of Food Quality prison
The aspects such as survey are respectively provided with extremely important application value.
Brief description of the drawings
Fig. 1 is AFB1 and AFM1 standard items chromatograms；
Fig. 2 is that liver adds AFB1 and AFM1 chromatograms；
Fig. 3 is that kidney adds AFB1 and AFM1 chromatograms；
Fig. 4 is that muscle adds AFB1 and AFM1 chromatograms；
Fig. 5 compares for different concussion time AFB1 and the AFM1 rate of recovery.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any limitation is not constituted to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications or replacement each fall within protection scope of the present invention.
The broiler chicken liver of embodiment 1, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography method
Detection
1st, experimental technique
The preparation of 1.1 reagents
PBS solution is prepared：Accurately weigh NaCl 8.0g, KCl 0.2g, KH2PO4 0.2g、Na2HPO4.12H2O 2.9g are molten
In about 800mL deionized waters, pH7.4 is adjusted, be settled to 1000mL, 4 DEG C of Refrigerator stores.
The preparation of AFB1 standard items：The 1mg packaging AFB1 standard items for taking Sigma-Aldrich are a, use color
Spectrum level methyl alcohol dissolves in brown volumetric flask and constant volume is to 10ml, the i.e. standard reserving solution of 100 μ g/ml, standby in -20 DEG C of refrigerators
With.
The preparation of Aflatoxins M1 standard items：Sigma-Aldrich, concentration are taken for 0.5 μ g/ml AFM1 standard liquids
1ml, with chromatographic grade acetonitrile in brown volumetric flask constant volume to 10ml, the i.e. standard reserving solution of 0.05 μ g/ml, in -20 DEG C of refrigerators
It is standby.
Two kinds of standard liquids are being diluted to required concentration with coordinative solvent respectively using preceding, standby in -4 DEG C of refrigerators.
1.2 experiment equipments prepare
1) Ultra Performance Liquid Chromatography instrument Acquity UPLC (Waters, US)
2) fluorescence detector (FLD, Waters, US)
3) chromatographic work stations of EMPOWER 3
4) Aspergillus flavus toxin immuno-affinity column (Beijing Huaan Mai Ke companies AFM1 immune affinity columns and VICAM companies of the U.S.
AFB1 immune affinity columns)
5) 0.2 μm of organic filter membrane (Waters, US)
6) pump stream crosshead (Agilent company)
7) MP2002 types electronic analytical balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.)
8) high speed rotating disintegrator (German Fritsch companies)
9) N-EVAPTM112 Nitrogen evaporators (Organomation Associates companies of the U.S.)
10) pH meter (plum Teller-support benefit Instrument Ltd.)
11) MS3 types vortex oscillator (IKA companies)
12) SHZ-C types water-bath constant temperature oscillator (Shanghai Pudong Physical and Optical Instruments Factory)
1.3 chromatographic conditions
Chromatographic column is Waters Acquity UPLC BEH C18, and specification is 50mm × 2.1mm × 1.7 μm；Flow velocity
0.2mL/min；Sample size is 10 μ L；Mobile phase is acetonitrile:Water (2:8)；25 DEG C of column temperature；Excitation wavelength 365nm, launch wavelength
435nm；Run time 4min.
The drafting of 1.4 standard curves
Two kinds of aflatoxin standard liquids are diluted to 6 series standard working solutions (table 1) with coordinative solvent respectively, by phase
The chromatographic peak area x of corresponding aflatoxin standard concentration y and aflatoxin does linear regression, is returned accordingly
Equation and coefficient correlation；The retention time and its coefficient of variation of aflatoxin standard items are investigated simultaneously.
The AFB1 of table 1 and M1 standard items series concentrations
1.5 rate of recovery and Precision Experiment
Recovery test：The accurate broiler chicken liver weighed not in contact with AFB1 (control group), kidney and muscle 2.00g, respectively
The aflatoxin standard solution of various concentrations is added, the addition concentration (table 2) of basic, normal, high three kinds of different contents is made, pressed
Processing method operation (1.7) of tissue samples.Each concentration repeats treatment simultaneously sample introduction 5 times, and the analysis of 10 μ L sample introductions is taken every time, according to
Obtain corresponding calculated by peak area recovery of standard addition.
The concentration of high, medium and low three kinds of aflatoxin in the blank tissue of table 2
Precision test：Accurately weigh control group broiler chicken liver, kidney and muscle 2.00g, be separately added into it is above-mentioned it is low, in,
The aflatoxin standard solution of 3 concentration high, (1.7) are operated by the processing method of tissue samples, and 10 μ L sample introductions are taken every time
Analysis, each concentration repeats treatment simultaneously sample introduction 5 times, calculates the in a few days coefficient of variation；METHOD FOR CONTINUOUS DETERMINATION 5 days, calculates the coefficient of variation in the daytime.
The measure of 1.6 sensitivity
The stock solution of various aflatoxin standard items is progressively diluted, sets up processing method to count with the softwares of EMPOWER 3
Signal to noise ratio is calculated, when being equal to 3 according to signal to noise ratio (S/N), test limit (LOD) is determined, when being equal to 10 according to signal to noise ratio (S/N), it is determined that
Quantitative limit (LOQ).
1.7 broiler chicken livers, kidney and in chicken AFB1 and M1 contents detection
Preparation of samples：Take the continuous contamination AFB1 sub-chronic intoxication broiler chicken (model group) of 28 days, Culling heart blood
After death, take out whole livers, kidney and both legs and chest muscle part muscle in place.
Sample extraction：Tissue is shredded in putting tissue mashing machine, after crushing, weigh 2 ± 0.02g of sample, add 20ml bis-
Chloromethanes, after 60Hz ultrasounds 10min, 400r/min shaking tables concussion 60min adds anhydrous sodium sulfate 2g, and (10000r/ is centrifuged
Min, 5min), supernatant 10ml is taken, nitrogen drying under 50 DEG C of water bath conditions adds 2ml methyl alcohol dissolved residues, adds pH7.4 PBS
Solution 13mL, by AFM1 immune affinity columns, 1-2 drops/sec of flow velocity, 10ml pure water rinsing immune affinity columns, press-in air discharge
Residual moisture in post, adds 1ml methyl alcohol wash-out, collects whole eluents, and nitrogen drying under 50 DEG C of water bath conditions adds 100 μ l
Trifluoroacetic acid, 200 μ l n-hexanes, 40 DEG C of water-baths derive 15min, nitrogen drying, 10% methanol aqueous solution under 50 DEG C of water bath conditions
Constant volume 1mL, loading.
It is consistent with the retention time of aflatoxin standard items according to aflatoxin to be detected, it is defined as target aspergillus flavus
Toxin.Linear regression is done according to aflatoxin standard concentration and chromatographic peak area, equation of linear regression is obtained, according to broiler chicken
Liver, kidney and the peak area of aflatoxin takes corresponding equation of linear regression to and quantified in muscle.
2nd, experimental result
The linear equation of 2.1 aflatoxin
The chromatogram of aflatoxin standard items is shown in Fig. 1；The linear equation of aflatoxin is shown in Table 3.
The linear equation of the AFB1 .M1 of table 3
The retention time and its coefficient of variation of 2.2 aflatoxin
The retention time and its coefficient of variation of aflatoxin standard items are shown in Table 4.
The retention time and its coefficient of variation of the AFB1 .M1 of table 4
2.3 rate of recovery and precision
According to table 5-10 results, broiler chicken liver, kidney and the average recovery rate of AFB1 is respectively in muscle
82.32%~85.56%, 85.34%~88.45% and 84.88%~89.73%；Precision respectively 1.47%~
4.45%th, 1.12%~3.54% and 2.01%~4.17%.Broiler chicken liver, kidney and in muscle Aflatoxins M1 it is average
The rate of recovery is respectively 92.17%~95.03%, 94.12%~97.21% and 95.32%~98.54%；Precision is respectively
1.83%~3.46%, 1.47%~3.81% and 1.96%~3.97%.
Liver addition aflatoxin chromatogram is shown in Fig. 2；Kidney addition aflatoxin chromatogram is shown in Fig. 3；Muscle is added
Aflatoxin chromatogram is shown in Fig. 4.
The AFB1 .M1 rate of recovery in the liver of table 5
The AFB1 .M1 rate of recovery in the kidney of table 6
The AFB1 .M1 rate of recovery in the muscle of table 7
AFB1 .M1 precision in the liver of table 8
AFB1 .M1 precision in the kidney of table 9
AFB1 .M1 precision in the muscle of table 10
2.4 test limits and quantitative limit
, in 0.008ng/mL, quantitative limit is in 0.02ng/mL for AFB1 test limit；Aflatoxins M1 test limit exists
0.003ng/mL, quantitative limit is at 0.01ng/mL (table 11).
The test limit and quantitative limit of the AFB1 .M1 of table 11
2.5 broiler chicken livers, kidney and in chicken AFB1 and M1 contents detection
Using detect simultaneously broiler chicken liver, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography
Method, detection AFB1 sub-chronic intoxication broiler chicken liver, kidney and in chicken AFB1 and M1 content.
In Normal group, two kinds of residuals of aflatoxin are not detected by three kinds of tissues, and in aspergillus flavus poison
In plain B1 sub-chronic intoxications group, two kinds of residuals of aflatoxin (table 12) are detected in three kinds of tissues.
The content of the AFB1 of table 12 and AFM1
The condition optimizing of experimental example 1 is tested
1st, the optimization of extracting mode
(1) optimization of extraction organic solvent
Take blank tissue, sample mark-on amount AFB1 is that 0.02 μ g/kg, AFM1 is 0.01 μ g/kg, respectively with dichloromethane,
70% methanol solution, 80% methanol solution, 90% methanol solution, pure methanol solution and 84% acetonitrile solution (percent by volume) are carried
Take, other extracting parameters and Ultra Performance Liquid Chromatography detect parameter with embodiment 1.Analyzed according to recovery of standard addition, it is molten when extracting
The recovery of standard addition of AFB1 and AFM1 is all remarkably higher than other Extraction solvents (table 13) when agent is dichloromethane.
Influence of the organic solvent of table 13 to AFB1 the and AFM1 rate of recovery (%)
Note：Aflatoxin of the same race when different solvents are extracted the rate of recovery compared with dichloromethane extraction recovery,
Capitalization A represents P<0.05, lowercase a represents P<0.01.
According to the property of aflatoxin, the organic solvent of general extraction mainly has methyl alcohol, dichloromethane and acetonitrile.This
Invention is compared to three kinds of organic solvents to the extraction effect of aflatoxin, wherein 80% methyl alcohol is to carry with 84% acetonitrile
The common solvent of aflatoxin in feed is taken, but for extracting the recovery of aflatoxin in the tissue such as liver, kidney, muscle
Rate is substantially less than dichloromethane.Therefore, the dichloromethane that selective extraction effect is good, the rate of recovery is high of the present invention is used as in Chicken Tissues
The Extraction solvent of AFB1 and AFM1.
(2) optimization of extraction time is shaken
Sample mark-on amount AFB1 is that 0.02 μ g/kg, AFM1 is 0.01 μ g/kg, and Extraction solvent is dichloromethane, and concussion is extracted
Time is respectively 30min, 50min, 60min, 70min, 90min, and the rotating speed of shaking table concussion is 400r/min (other extracting parameters
And Ultra Performance Liquid Chromatography detects parameter with embodiment 1), inquire into influence of the concussion time to recovery of standard addition.
From table 14 and Fig. 5, the extraction recovery of concussion 30min and 50min is significantly or pole is substantially less than 60min, shakes
The extraction recovery for swinging 70min or 90min is not notable with concussion 60min differences.Therefore, the present invention is at preferred concussion extraction time
60min。
Table 14 shakes influence of the time to AFB1 the and AFM1 rate of recovery (%)
Note:The rate of recovery of the aflatoxin of the same race under the different concussion times respectively with concussion 60min groups rate of recovery phase
Than capitalization difference represents P<0.05, lowercase difference represents P<0.01.
(3) investigation of immune affinity column
By aflatoxin standard items by AFM1 immune affinity columns or AFB1 immune affinity columns, compare the species of affinity column
Influence to the rate of recovery.Result shows that the immune affinity column of Aflatoxins M1 is very high to the rate of recovery of AFM1 and AFB1, and
AFB1 immune affinity columns can not adsorb AFM1 (table 15).It is therefore preferable that AFM1 immune affinity columns.
Influence of the different immune affinity columns of table 15 to AFB1 the and AFM1 rate of recovery (%)
(4) constant volume solution
Investigation is respectively 10%, 20%, 50%, 70%, 100% with methanol aqueous solution percent by volume respectively；Acetonitrile water
Solution proportion is 10%, 20%, 50%, 70%, 100% (percent by volume)；The last drying sample of dissolving constant volume, analysis is not
Influence with constant volume solution to sample chromatogram separating effect after sample introduction (Ultra Performance Liquid Chromatography detects parameter with embodiment 1).
Result shows that organic phase selects methyl alcohol, and ratio, at 10%, chromatographic peak peak type is good, and solvent peak is to AFM1 peak types
Interference is small, and peak area is big.Therefore preferably 10% methanol aqueous solution constant volume.
2nd, chromatographic condition optimization
(1) Optimization of mobile phase
Flow velocity 0.2ml/min, the μ l of sample size 10,25 DEG C of column temperature；Standard concentration AFB1 is 0.02ng/ml, and AFM1 is
0.01ng/ml；Analysis mobile phase acetonitrile and water volume ratio are respectively 15:85、20:80、25:75 and 30:70 couples of AFB1 and AFM1
The influence (other Ultra Performance Liquid Chromatographies detect parameter with embodiment 1) of peak area.
Result shows, acetonitrile:Water is 20:Peak area under the conditions of 80 mobile phases is noticeably greater than under the conditions of other mobile phases
Peak area (table 16).Therefore, the preferred acetonitrile of mobile phase of the present invention:Water is 20:80.
The influence of the mobile phase acetonitrile of table 16 and water ratio to AFB1 and AFM1 peak areas
Note:The peak that peak area of the aflatoxin of the same race under the conditions of different mobile phases is organized with 20+80 (acetonitrile+water) respectively
Area is compared, and capitalization A represents P<0.05, lowercase a represents P<0.01.
(2) flowing rate
The μ l of sample size 10,25 DEG C of column temperature, mobile phase acetonitrile：Water is 20：80；Standard concentration AFB1 is 0.02ng/ml,
AFM1 is 0.01ng/ml；Influence (other ultra high efficiency liquid of analysis flow velocity when being respectively 0.1,0.2 and 0.3ml/min to peak area
Phase chromatogram detects parameter with embodiment 1).
Influence of the flow velocity of table 17 to AFB1 and AFM1 peak areas
Note:Peak area of the aflatoxin of the same race under the conditions of different in flow rate respectively with the peak area phase of 0.2ml/min groups
Than capitalization A represents P<0.05, lowercase a represents P<0.01.
Result shows that appearance time is late when flow velocity is 0.1ml/min, and peak type is low flat；It is 0.3ml/min in flow velocity
When, appearance time too early, is separated not thorough with solvent peak；Peak type is good when flow velocity is 0.2ml/min, and peak area compares with other treatment
Compared with significant difference (P<0.01) (table 17), chromatogram peak energy is preferably separated.Therefore, preferable flow rate of the present invention is 0.2ml/
min。
(3) column temperature optimization
The μ l of sample size 10, mobile phase acetonitrile：Water is 20：80, flow velocity 0.2ml/min；Standard concentration AFB1 is 0.02ng/
Ml, AFM1 are 0.01ng/ml；Analysis column temperature is respectively 20,25,30 DEG C of influence (other Ultra Performance Liquid Chromatographies to peak area
Detection parameter is with embodiment 1).
Influence of the column temperature of table 18 to AFB1 and AFM1 peak areas
Note:Peak area of the aflatoxin of the same race under the conditions of different column temperatures respectively compared with 25 DEG C of peak areas of group, greatly
Female A that writes represents P<0.05, lowercase a represents P<0.01.
Result shows that when column temperature is 25 DEG C, good separating effect, peak type is good, and peak area is big, with other treatment comparing differences
Significantly (P<0.01) (table 18).Therefore preferably column temperature is 25 DEG C.
(4) investigation of Detection wavelength
Excitation wavelength is set to 360nm, launch wavelength is scanned in 390~400nm, comprehensive AFB1, AFM1 maximum absorption waves
Long, 435nm is used as final launch wavelength for selection.
Launch wavelength is set to 435nm, excitation wavelength is scanned in 300~400nm, comprehensive AFB1, AFM1 maximum absorption waves
Long, 365nm is used as final excitation wavelength for selection.
Claims (10)
1. a kind of at the same detect broiler chicken liver, kidney and in chicken AFB1 and M1 contents Ultra Performance Liquid Chromatography side
Method, it is characterised in that comprise the following steps：
(1) AFB1 and M1 in liver, kidney or the Chicken Tissues of broiler chicken to be detected are extracted, extract solution is obtained；(2)
Extract solution is purified, is derived；(3) qualitative, quantitative measure is carried out using Ultra Performance Liquid Chromatography.
2. according to the Ultra Performance Liquid Chromatography method described in claim 1, it is characterised in that step (1) is described to extract to be detected
AFB1 and M1 in the liver of broiler chicken, kidney or Chicken Tissues include：By the liver of broiler chicken to be detected, kidney or chicken
Meat tissue crush, add Extraction solvent, it is sonicated after, shaking table concussion, be subsequently adding anhydrous sodium sulfate, be centrifuged, take supernatant.
3. according to the Ultra Performance Liquid Chromatography method described in claim 2, it is characterised in that the Extraction solvent is selected from dichloromethane
Any one in alkane, 70%-100% methanol solutions or 84% acetonitrile solution, preferably dichloromethane；
It is furthermore preferred that being counted according to g/ml, the liver of the broiler chicken to be detected, kidney or Chicken Tissues：Extraction solvent=1：10.
4. according to the Ultra Performance Liquid Chromatography method described in claim 2, it is characterised in that the time of the ultrasound is 10min；
The power of ultrasound is 60Hz；
The time of the shaking table concussion is 30-90min, preferably 60min；The rotating speed of the shaking table concussion is 400r/min.
5. it is described to be checked according to the Ultra Performance Liquid Chromatography method described in claim 2, it is characterised in that according to mass ratio meter
Survey liver, kidney or the Chicken Tissues of broiler chicken：Anhydrous sodium sulfate=1：1.
6. according to the Ultra Performance Liquid Chromatography method described in claim 1, it is characterised in that step (2) purification includes：Will
Extract solution collects eluent by Aspergillus flavus toxin immuno-affinity column；
Step (2) the derivative method is trifluoroacetic acid pre-column derivatization；Product after derivative is used through nitrogen drying, constant volume
In loading.
7. according to the Ultra Performance Liquid Chromatography method described in claim 6, it is characterised in that the Aspergillus flavus toxin immuno is affine
Post is selected from any one in AFM1 immune affinity columns or AFB1 immune affinity columns, preferably AFM1 immune affinity columns；
The solution of the constant volume is 10%-100% methanol aqueous solutions or 10%-100% acetonitrile solutions, preferably 10% methyl alcohol
The aqueous solution.
8. according to the Ultra Performance Liquid Chromatography method described in claim 1, it is characterised in that step (3) the ultra high efficiency liquid phase
The condition of chromatogram includes：Chromatographic column is Waters Acquity UPLC BEHC18, and specification is 50mm × 2.1mm × 1.7 μm；Stream
Fast 0.1-0.3mL/min；Sample size is 10 μ L；Mobile phase is acetonitrile：Water；20-30 DEG C of column temperature；Excitation wavelength 365nm, transmitted wave
435nm long.
9. according to the Ultra Performance Liquid Chromatography method described in claim 8, it is characterised in that：According to volume basis, the flowing
It is mutually acetonitrile：Water=15-30：70-85；Preferably, the mobile phase is acetonitrile：Water=20：80；
The column temperature is 25 DEG C；The flow velocity is 0.2mL/min.
10. according to the Ultra Performance Liquid Chromatography method described in claim 1, it is characterised in that step (3) qualitative, quantitative determines bag
Include：It is consistent with the retention time of aflatoxin standard items according to aflatoxin to be detected, it is defined as target aflatoxin；
Linear regression is done according to aflatoxin standard concentration and chromatographic peak area, equation of linear regression is obtained；Will be to be detected
The liver of broiler chicken, kidney or the peak area of aflatoxin brings equation of linear regression into Chicken Tissues, calculate target aspergillus flavus
The respective concentration of toxin.
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