CN106290689B - The method that eutectic solvent extraction liquid chromatography quickly determines aflatoxin B1, Basic Orange II and basic flavine O - Google Patents
The method that eutectic solvent extraction liquid chromatography quickly determines aflatoxin B1, Basic Orange II and basic flavine O Download PDFInfo
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- CN106290689B CN106290689B CN201610853014.7A CN201610853014A CN106290689B CN 106290689 B CN106290689 B CN 106290689B CN 201610853014 A CN201610853014 A CN 201610853014A CN 106290689 B CN106290689 B CN 106290689B
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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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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- 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
- G01N30/02—Column chromatography
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- G01N2030/062—Preparation extracting sample from raw material
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- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention provides the method that a kind of eutectic solvent extraction liquid chromatography quickly determines aflatoxin B1, Basic Orange II and basic flavine O, it comprises the following steps, first prepare the solution of aflatoxin B1, Basic Orange II and basic flavine O, it is diluted to the standard working solution of various concentrations, and the liquid chromatogram of examination criteria working solution, and make calibration curve；Eutectic solvent is prepared, eutectic solvent includes hydrogen bond acceptor compounds and hydrogen bond donor compound；Seasoning oil samples are weighed, eutectic solvent and alkane reagent is added, is extracted, oil reservoir above is removed after centrifugation, residue is detected to obtain the chromatogram of sample after being washed with alkane using liquid chromatograph；By the chromatogram of sample compared with the chromatogram of standard working solution it is qualitative, quantified further according to the concentration on the standard curve corresponding to peak area.Technical scheme is practical, simple to operate, it is not necessary to which complicated pretreatment process, experimental cost are low.
Description
Technical field
The invention belongs to non-food coloring detection technique field in food, more particularly to a kind of eutectic solvent extraction liquid phase
The method that chromatography quickly determines aflatoxin B1, Basic Orange II and basic flavine O.
Background technology
Basic Orange II and basic flavine O are aromatic amine alkalescence industrial dye, are mainly used in fiber crops, paper, leather, straw plaited
The dyeing of product, artificial silk etc..Basic Orange II and basic flavine O have slight stimulation to skin and mucosa, can cause conjunctivitis, dermatitis and
Upper respiratory tract irritation, human contact or sucks and can cause poisoning, and is difficult katabolism, and both materials are respectively provided with cause
Cancer and mutagenesis property.Sanitary standard regulation is applicable according to the food additives of GB 2760, Basic Orange II and basic flavine O are all tight
Taboo uses as food additives.Because under the conditions of neutral or meta-alkalescence, Basic Orange II and basic flavine O adsorb with protein
Stronger, fugitive color, is not easier to dye in food than other water-soluble dyes such as lemon yellow, sunset yellow, therefore, some
Illegal retailer often uses it for the dyeing of food, makes product color vivid, cheats consumer, endangers the health of consumer.
GB 35/T 897-2009《The measure of food neutral and alkali orange, basic flavine O and alkaline pink T contents》In define
Basic Orange II and basic flavine O detection method, this method exist pretreatment process very complicated, organic solvent usage amount compared with
More, many defects such as time-consuming, and this method is primarily directed to solid sample, for this kind of matrix of flavored oils and impurity
More complicated sample does not apply to simultaneously.
Aflatoxin (AFT) is one of generally acknowledged natural strong carcinogen, wherein with aflatoxin B1(AFTB1) it is malicious
Property it is maximum, they are normally present in various animal feeds and human food.China mycotoxin limit standard GB 2761《Food
Mycotoxin is limited the quantity in safe national standard food》Aflatoxin B is defined for different food products kind1Limit index.
GB/T 18979-2003《The measure immunoaffinity chromatography purification high performance liquid chromatography of aflatoxin in food
And fluorimetry》With GB/T 5009.23-2006《Aflatoxin B in food1、B2、G1、G2Measure》In define
Aflatoxin B1Detection method, there is pretreatment process very complicated in this method, organic solvent usage amount is more, time-consuming
Etc. many defects.
The content of the invention
For above technical problem, the invention discloses a kind of eutectic solvent extraction liquid chromatography quickly to determine yellow song
Mould toxin B1, Basic Orange II and basic flavine O method, this method is simple to operate, and detection is quick, and cost is cheap.
On the other hand, the technical solution adopted by the present invention is：
A kind of eutectic solvent extraction liquid chromatography quickly determines aflatoxin B1, Basic Orange II and basic flavine O
Method, it comprises the following steps：
Step S1：The hybrid standard liquid of Basic Orange II and basic flavine O is prepared, and is diluted to the standard work of various concentrations
Liquid, liquid chromatograph is respectively adopted and is detected to obtain the liquid of the Basic Orange II of various concentrations and basic flavine O standard working solution
Phase chromatogram, the calibration curve of Basic Orange II and basic flavine O is made according to the peak area of liquid chromatogram and corresponding concentration；
Aflatoxin B1 titer is prepared, and is diluted to the standard working solution of the aflatoxin B1 of various concentrations respectively, is adopted respectively
Detected to obtain the liquid chromatogram of various concentrations aflatoxin B1 standard working solution with liquid chromatograph, according to liquid phase color
The peak area of spectrogram and corresponding concentration make the calibration curve of aflatoxin B1；
Step S2：Eutectic solvent is prepared, the eutectic solvent includes hydrogen bond acceptor compounds and hydrogen bond donor chemical combination
Thing；
Step S3：Seasoning oil samples are weighed, eutectic solvent and alkane reagent described in step S2 is added, carries out concussion extraction
Take, oil reservoir above is removed after centrifugation, residue is detected to obtain the chromatogram of sample using liquid chromatograph；Wherein, institute
The volume ratio for stating eutectic solvent and alkane reagent is more than 0.0025；Wherein preferable, the residue is adopted after being washed with alkane
Detected with liquid chromatograph, the purpose of washing is in order to which oil removing removal of impurities is more thorough；
Step S4：By the liquid chromatogram of the chromatogram of sample and Basic Orange II and basic flavine O standard working solution and
The liquid chromatogram of aflatoxin B1 standard working solution, which is compared, to be determined whether containing Basic Orange II, basic flavine O and Huang
Aspertoxin B1, if it is determined that containing Basic Orange II and basic flavine O, further according to the mark corresponding to the chromatogram peak area of sample
Concentration on directrix curve determines the content of sample neutral and alkali orange II and basic flavine O；If it is determined that contain aflatoxin B1, then
The concentration on standard curve according to corresponding to the chromatogram peak area of sample determines the content of aflatoxin B1 in sample.
Using this technical scheme, the Basic Orange II and basic flavine O in seasoning oil samples, profit are extracted using eutectic solvent
The purpose that complicated matrix can reach sample purification is removed with alkanes reagent, then by machine testing on eutectic solvent, is realized
Quick detection, step is simple, without the sample pretreatment process of complexity, reduces the use of solvent, while eliminate seasoning again
The interference of complex matrices in oil, ensure that the accuracy of testing result.
As a further improvement on the present invention, the step S1 also includes：Aflatoxin B1 titer is prepared, and respectively
Be diluted to the standard working solution of the aflatoxin B1 of various concentrations, be respectively adopted liquid chromatograph detected to obtain it is different dense
The liquid chromatogram of aflatoxin B1 standard working solution is spent, is made according to the peak area of liquid chromatogram and corresponding concentration yellow
Aspertoxin B1 calibration curve；
The step S4 also includes：The liquid chromatogram of the chromatogram of sample and aflatoxin B1 standard working solution is entered
Row is relatively determined whether containing aflatoxin B1, if it is determined that containing aflatoxin B1, further according to the chromatogram peak out of sample
The concentration on standard curve corresponding to area determines the content of aflatoxin B1 in sample.
Using this technical scheme, Basic Orange II and basic flavine O non-food coloring and aflatoxin can be carried out simultaneously
B1 detection, processing is simple, operational version, examines quick, the shortcomings that overcoming existing detection method and deficiency.
As a further improvement on the present invention, the hydrogen bond donor compound is at least one of urea and alcohols, institute
The mol ratio for stating hydrogen bond acceptor compounds and hydrogen bond donor compound is 1：(1~3).
Preferably, the dosage of the eutectic solvent is according to every gram of 0.05~5.0mL of seasoning oil samples.
As a further improvement on the present invention, the hydrogen bond acceptor compounds are glycine betaine, the glycine betaine and the hydrogen
The mol ratio of key compound donator is 1:1、1:2 or 1:3.
As a further improvement on the present invention, the hydrogen bond donor compound is glycerine.
As a further improvement on the present invention, the alkane reagent is included in n-hexane, hexamethylene, octane, heptane extremely
Few one kind.
As a further improvement on the present invention, the volume ratio of eutectic solvent and the alkane reagent is 0.5~5.
As a further improvement on the present invention, the alkane reagent is n-hexane or normal heptane.
As a further improvement on the present invention, in step S3, the mode of the extraction is shaking table concussion extraction or ultrasonic wave
Extraction, the Extracting temperature is at 10-50 DEG C.Preferably, the mode of the extraction is shaking table concussion extraction.
As a further improvement on the present invention, in step S3, residue washs the oil for removing residual with alkane, then will be low
Congruent melting solvent directly goes up machine or constant volume to upper machine testing after required volume.
Compared with prior art, beneficial effects of the present invention are：
First, technical scheme is practical, for the viscosity such as chilli oil, chilly oil, Arnotto orange oil it is big,
The complicated seasoning oil samples of matrix, can carry out the accurate detection of Basic Orange II, basic flavine O and aflatoxin B1.
Second, technical scheme is simple to operate, cost is low, present invention only requires simple sample extraction process,
Complicated pretreatment process is not needed, the use of organic solvent can be saved by extracting eutectic solvent used, be greatly reduced
Experimental cost.
3rd, technical scheme detection is quick, because this method can save time for sample pretreatment, contracts significantly
Duan Liao flavored oils neutral and alkali orange II, basic flavine O and aflatoxin B1Time required for detection.
4th, recovery of standard addition is high, and detection limit is low, and eutectic solvent can effectively extract Basic Orange from flavored oils
II, basic flavine O and aflatoxin B1, while alkanes reagent can remove the oil matrix of complexity, avoid impurity from disturbing,
Therefore the sufficiently high rate of recovery and relatively low detection limit can be ensured, the rate of recovery of sample respectively 82.3%-101%,
In the range of 81.6%-103% and 75.6%-108%, detection limit is respectively up to 0.02mg/kg, 0.02mg/kg and 1.0 μ g/kg.
Brief description of the drawings
Fig. 1 is two kinds of pigment mixing of the Basic Orange II that the concentration of an embodiment of the present invention is 0.1 μ g/L and basic flavine O
Standard working solution chromatogram.
Fig. 2 is the aflatoxin B1 standard working solution chromatogram that an embodiment of the present invention concentration is 0.5 μ g/L.
Fig. 3 is the Basic Orange II standard working solution calibration curves of an embodiment of the present invention.
Fig. 4 is the basic flavine O standard working solution calibration curve of an embodiment of the present invention.
Fig. 5 is the aflatoxin B of an embodiment of the present invention1Standard working solution calibration curve.
Embodiment
The present invention is made with reference to Figure of description and specific embodiment and further being elaborated, the embodiment
It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Material, reagent, instrument used in following embodiments
Equipment etc., it is commercially available unless otherwise specified.
Methanol：Chromatographically pure, it is purchased from Merck ＆ Co., Inc.；Acetonitrile：Chromatographically pure, it is purchased from Merck ＆ Co., Inc.；N-hexane：Chromatographically pure, it is purchased from
Merck ＆ Co., Inc.；Normal heptane：Chromatographically pure, it is purchased from Merck ＆ Co., Inc.；Ammonium acetate：Analyze it is pure, be purchased from Town in Shanghai spectrum, glacial acetic acid：Analyze it is pure,
Shanghai Ling Feng；Glycine betaine：Analyze pure, Aladdin；Urea：Analyze pure, Aladdin；Glycerine：Analyze pure, Shanghai Ling Feng；The third two
Alcohol：Analyze pure, Shanghai Ling Feng；Water is ultra-pure water.
Basic Orange II standard items：Purity 95.00%, purchased from Sigma-Aldrich.
Basic flavine O standard items：Purity 86.00%, purchased from Sigma-Aldrich.
Aflatoxin B1Standard items：Purity 100.0%, purchased from lark prestige.
High performance liquid chromatograph：It is equipped with quaternary gradient pump, degasser, microsyringe, UV-detector and fluorescence inspection
Survey device.
Basic Orange II and basic flavine O liquid phase chromatogram condition parameter are as follows：
Chromatographic column：Agilent 5TC-C18,150 × 4.6mm
Column temperature：25℃
Detection wavelength：450nm
Ammonium acetate buffer solution：0.77g ammonium acetate solids are weighed, are dissolved in water, are transferred in 1000mL volumetric flasks, then are added
Enter 1.2mL glacial acetic acid, be settled to scale with ultra-pure water, ultrasound removes bubble, filters and removes solid impurity.
Sample size：25μL.
Flow velocity：1.0mL/min.
Condition of gradient elution：Mobile phase A is acetonitrile, and Mobile phase B is ammonium acetate buffer solution.
The concrete condition of mobile phase is as shown in table 1.
Table 1
Aflatoxin B1Liquid phase chromatogram condition parameter is as follows：
Chromatographic column：Agilent 5TC-C18,150 × 4.6mm
Column temperature：40℃
Detection wavelength：Excitation wavelength 360nm, launch wavelength 420nm
Sample size：20μL
Flow velocity：0.8mL/min
Isocratic condition：Methanol：Water=45：55
Post-column derivation condition：0.05% iodine solution, flow velocity 0.2mL/min, 70 DEG C of reaction temperature
Follow the steps below detection：
Step S1：The hybrid standard liquid of Basic Orange II and basic flavine O is prepared, and is diluted to the standard work of various concentrations
Liquid, liquid chromatograph is respectively adopted and is detected to obtain the liquid of the Basic Orange II of various concentrations and basic flavine O standard working solution
Phase chromatogram, the calibration curve of Basic Orange II and basic flavine O is made according to the peak area of liquid chromatogram and corresponding concentration；
And aflatoxin B1 titer is prepared, and the standard working solution of the aflatoxin B1 of various concentrations is diluted to respectively, respectively
Detected to obtain the liquid chromatogram of various concentrations aflatoxin B1 standard working solution using liquid chromatograph, according to liquid phase
The peak area of chromatogram and corresponding concentration make the calibration curve of aflatoxin B1；
Step S2：Prepare eutectic solvent.
Eutectic solvent DES-1 preparation：20g glycine betaines and 15.72g glycerine are weighed in 100mL reaction bulbs, is risen
It is standby to be cooled to room temperature to 90 DEG C and untill stirring to clarify for temperature.
Eutectic solvent DES-2 preparation：20g glycine betaines and 20.51g urea are weighed in 100mL reaction bulbs, heating
Untill to 90 DEG C and stirring to clarify, it is standby to be cooled to room temperature.
Eutectic solvent DES-3 preparation：20g glycine betaines and 31.79g ethylene glycol are weighed in 100mL reaction bulbs, is risen
It is standby to be cooled to room temperature to 90 DEG C and untill stirring to clarify for temperature.
Step S3：Seasoning oil samples are weighed, eutectic solvent and alkane reagent described in step S2 is added, carries out concussion extraction
Take, oil reservoir above is removed after centrifugation, residue is detected to obtain the color of sample after being washed with alkane using liquid chromatograph
Spectrogram；Wherein, the volume ratio of eutectic solvent and the alkane reagent is more than 0.0025；
Step S4：By the liquid chromatogram of the chromatogram of sample and Basic Orange II and basic flavine O standard working solution and
The liquid chromatogram of aflatoxin B1 standard working solution, which is compared, to be determined whether containing Basic Orange II and basic flavine O, such as
Fruit determines to contain Basic Orange II and basic flavine O, further according to dense on the standard curve corresponding to the chromatogram peak area of sample
Degree determines the content of sample neutral and alkali orange II and basic flavine O；By the chromatogram of sample and aflatoxin B1 standard working solution
Liquid chromatogram be compared and determine whether containing aflatoxin B1, if it is determined that containing aflatoxin B1, further according to
The concentration on standard curve corresponding to the chromatogram peak area of sample determines the content of aflatoxin B1 in sample.Concentration is
0.1 μ g/L Basic Orange II and two kinds of pigment hybrid standard working solution chromatograms of basic flavine O is as shown in Figure 1；Concentration is 0.5 μ
G/L aflatoxin B1 standard working solution chromatogram is as shown in Figure 2；Basic Orange II standard working solutions calibration curve such as Fig. 3 institutes
Show；Basic flavine O standard working solution calibration curve is as shown in Figure 4；Aflatoxin B1Standard working solution calibration curve such as Fig. 5 institutes
Show.
Embodiment 1
Sample：Chilly oil 1g is weighed in 15mL centrifuge tubes, adds 0.5mL eutectic solvent DES-1 and 8mL n-hexanes,
Shaking table concussion extraction 10 minutes, 7500r/min is centrifuged 2 minutes, and the oil reservoir drawn above with plastic suction pipe discards, and adds 4mL
N-hexane, concussion washing 1 minute, 7500r/min is centrifuged 2 minutes, and oil reservoir above is siphoned away with plastic suction pipe to be discarded.What is remained is low
Congruent melting solvent DES-1 part methanol constant volumes are vortexed and mixed, cross 0.22 μm of organic filter membrane, examination with computer to 2.0mL.
Mark-on sample 1：Chilly oil 1g is weighed in 15mL centrifuge tubes, addition Basic Orange II, basic flavine O and aspergillus flavus
Toxin B1Concentration level be 2.0mg/kg, add 0.5mL eutectic solvent DES-1 and 8mL n-hexanes, shaking table concussion extraction
10 minutes, 7500r/min was centrifuged 2 minutes, and the oil reservoir drawn above with plastic suction pipe discards, and adds 4mL n-hexanes, concussion is washed
Wash 1 minute, 7500r/min is centrifuged 2 minutes, and oil reservoir above is siphoned away with plastic suction pipe to be discarded.The eutectic solvent DES-1 of residual
Part methanol constant volume is vortexed and mixed, cross 0.22 μm of organic filter membrane, examination with computer to 2.0mL.
By the chromatogram of sample and Basic Orange II and basic flavine O standard working solution chromatogram and aflatoxin
The liquid chromatogram of B1 standard working solutions, which is compared, to be determined whether containing Basic Orange II and basic flavine O or simultaneously containing Huang
Aspertoxin B1, sample neutral and alkali orange is determined further according to the concentration on the standard curve corresponding to the chromatogram peak area of sample
II, basic flavine O, the content of aflatoxin B1, obtained result are as shown in table 2.
The testing result table of the embodiment 1 of table 2
Embodiment 2
Sample：Chilli oil 1g is weighed in 15mL centrifuge tubes, adds 0.3mL eutectic solvent DES-2 and 5mL normal heptanes,
Shaking table concussion extraction 10 minutes, 7500r/min is centrifuged 2 minutes, and the oil reservoir drawn above with plastic suction pipe discards, and adds 5mL
Normal heptane, concussion washing 1 minute, 7500r/min is centrifuged 2 minutes, and oil reservoir above is siphoned away with plastic suction pipe to be discarded.What is remained is low
Congruent melting solvent DES-2 part methanol constant volumes are vortexed and mixed, cross 0.22 μm of organic filter membrane, examination with computer to 2.0mL.
Mark-on sample 2：Chilli oil 1g is weighed in 15mL centrifuge tubes, addition Basic Orange II, basic flavine O and aspergillus flavus
Toxin B1Concentration level be 2.0mg/kg, add 0.3mL eutectic solvent DES-2 and 5mL normal heptanes, shaking table concussion extraction
10 minutes, 7500r/min was centrifuged 2 minutes, and the oil reservoir drawn above with plastic suction pipe discards, and adds 5mL normal heptanes, concussion is washed
Wash 1 minute, 7500r/min is centrifuged 2 minutes, and oil reservoir above is siphoned away with plastic suction pipe to be discarded.The eutectic solvent DES-2 of residual
Part methanol constant volume is vortexed and mixed, cross 0.22 μm of organic filter membrane, examination with computer to 2.0mL.
By the chromatogram of sample and Basic Orange II and basic flavine O standard working solution chromatogram and aflatoxin
Simultaneously whether the liquid chromatogram of B1 standard working solutions is compared determines whether containing Basic Orange II and basic flavine O, and
Containing aflatoxin B1, alkali in sample is determined further according to the concentration on the standard curve corresponding to the chromatogram peak area of sample
Property orange II, basic flavine O, the content of aflatoxin B1, obtained result are as shown in table 3.
The testing result table of the embodiment 2 of table 3
Embodiment 3
Sample：Curcuma oil 1g is weighed in 15mL centrifuge tubes, adds 0.5mL eutectic solvent DES-3 and 5mL normal heptanes,
Shaking table concussion extraction 10 minutes, 7500r/min is centrifuged 2 minutes, and the oil reservoir drawn above with plastic suction pipe discards, and adds 5mL
Normal heptane, concussion washing 1 minute, 7500r/min is centrifuged 2 minutes, and oil reservoir above is siphoned away with plastic suction pipe to be discarded.What is remained is low
Congruent melting solvent DES-3 part methanol constant volumes are vortexed and mixed, cross 0.22 μm of organic filter membrane, examination with computer to 2.0mL.
Mark-on sample 3：Curcuma oil 1g is weighed in 15mL centrifuge tubes, addition Basic Orange II, basic flavine O and aspergillus flavus
Toxin B1Concentration level be 2.0mg/kg, add 0.5mL eutectic solvent DES-3 and 5mL normal heptanes, shaking table concussion extraction
10 minutes, 7500r/min was centrifuged 2 minutes, and the oil reservoir drawn above with plastic suction pipe discards, and adds 5mL normal heptanes, concussion is washed
Wash 1 minute, 7500r/min is centrifuged 2 minutes, and oil reservoir above is siphoned away with plastic suction pipe to be discarded.The eutectic solvent DES-3 of residual
Part methanol constant volume is vortexed and mixed, cross 0.22 μm of organic filter membrane, examination with computer to 2.0mL.
By the chromatogram of sample and Basic Orange II and basic flavine O standard working solution chromatogram and aflatoxin
The liquid chromatogram of B1 standard working solutions, which is compared, to be determined whether containing Basic Orange II and basic flavine O or simultaneously containing Huang
Aspertoxin B1, sample neutral and alkali orange is determined further according to the concentration on the standard curve corresponding to the chromatogram peak area of sample
II, basic flavine O, the content of aflatoxin B1, obtained result are as shown in table 4.
The testing result table of the embodiment 3 of table 4
By testing repeatedly, the rate of recovery of sample containing Basic Orange II, basic flavine O, aflatoxin B1 exists respectively
In the range of 82.3%-101%, 81.6%-103% and 75.6%-108%, Basic Orange II, basic flavine O, aflatoxin B1
Detection limit respectively up to 0.02mg/kg, 0.02mg/kg and 1.0 μ g/kg.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to is assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (10)
1. a kind of eutectic solvent extraction liquid chromatography quickly determines aflatoxin B1, Basic Orange II and basic flavine O
Method, it is characterised in that：It comprises the following steps：
Step S1：The hybrid standard liquid of Basic Orange II and basic flavine O is prepared, and is diluted to the standard working solution of various concentrations,
Liquid chromatograph is respectively adopted to be detected to obtain the liquid phase color of the Basic Orange II of various concentrations and basic flavine O standard working solution
Spectrogram, the calibration curve of Basic Orange II and basic flavine O is made according to the peak area of liquid chromatogram and corresponding concentration；Prepare
Aflatoxin B1 titer, and the standard working solution of the aflatoxin B1 of various concentrations is diluted to respectively, liquid is respectively adopted
Chromatography is detected to obtain the liquid chromatogram of various concentrations aflatoxin B1 standard working solution, according to liquid chromatogram
Peak area and corresponding concentration make aflatoxin B1 calibration curve；
Step S2：Eutectic solvent is prepared, the eutectic solvent includes hydrogen bond acceptor compounds and hydrogen bond donor compound；
Step S3：Seasoning oil samples are weighed, eutectic solvent and alkane reagent described in step S2 is added, carries out concussion extraction,
Oil reservoir above is removed after centrifugation, residue is detected to obtain the chromatogram of sample using liquid chromatograph；Wherein, it is described low
The volume ratio of congruent melting solvent and alkane reagent is more than 0.0025；
Step S4：By the chromatogram of sample and Basic Orange II and basic flavine O standard working solution liquid chromatogram and yellow song
The liquid chromatogram of mould toxin B1 standard working solutions, which is compared, to be determined whether containing Basic Orange II, basic flavine O, aspergillus flavus poison
Plain B1, if it is determined that containing Basic Orange II and basic flavine O, further according to the standard curve corresponding to the chromatogram peak area of sample
On concentration determine the content of sample neutral and alkali orange II and basic flavine O；If it is determined that contain aflatoxin B1, further according to sample
The concentration on standard curve corresponding to the chromatogram peak area of product determines the content of aflatoxin B1 in sample；
Wherein, hydrogen bond acceptor compounds are glycine betaine in eutectic solvent, the hydrogen bond donor compound be glycerine, urea or
Ethylene glycol；The glycine betaine and the mol ratio minimum 1 of the hydrogen bond donor compound：1, up to 1：3.
2. eutectic solvent extraction liquid chromatography according to claim 1 quickly determines aflatoxin B1, Basic Orange
II and basic flavine O method, it is characterised in that：The dosage of the eutectic solvent is according to every gram of seasoning oil samples 0.05 ~ 5.0
mL。
3. eutectic solvent extraction liquid chromatography according to claim 2 quickly determines aflatoxin B1, Basic Orange
II and basic flavine O method, it is characterised in that：The mol ratio of the glycine betaine and the hydrogen bond donor compound is 1:1、1:
2 or 1:3.
4. eutectic solvent extraction liquid chromatography according to claim 3 quickly determines aflatoxin B1, Basic Orange
II and basic flavine O method, it is characterised in that：The hydrogen bond donor compound is urea.
5. the eutectic solvent extraction liquid chromatography according to claim 1 ~ 4 any one quickly determines aflatoxin
B1, Basic Orange II and basic flavine O method, it is characterised in that：The alkane reagent includes n-hexane, hexamethylene, octane, heptan
At least one of alkane.
6. eutectic solvent extraction liquid chromatography according to claim 5 quickly determines aflatoxin B1, Basic Orange
II and basic flavine O method, it is characterised in that：The volume ratio of eutectic solvent and the alkane reagent is 0.5 ~ 5.
7. eutectic solvent extraction liquid chromatography according to claim 6 quickly determines aflatoxin B1, Basic Orange
II and basic flavine O method, it is characterised in that：The alkane reagent is n-hexane or normal heptane.
8. the eutectic solvent extraction liquid chromatography according to claim 1 ~ 4 any one quickly determines aflatoxin
B1, Basic Orange II and basic flavine O method, it is characterised in that：In step S3, the mode of the extraction is shaking table concussion extraction
Or ultrasonic wave extraction, the temperature of the extraction is at 10-50 DEG C.
9. the eutectic solvent extraction liquid chromatography according to claim 1 ~ 4 any one quickly determines aflatoxin
B1, Basic Orange II and basic flavine O method, it is characterised in that：In step S3, residue is washed with alkane and removes residual
Oil, machine or constant volume are then directly gone up to upper machine testing after required volume.
10. the eutectic solvent extraction liquid chromatography according to claim 1 ~ 4 any one quickly determines aspergillus flavus poison
Plain B1, Basic Orange II and basic flavine O method, it is characterised in that：In step S3, the residue is adopted again after being washed with alkane
Tested with liquid chromatograph.
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