CN102288716A - Method for testing residual canthaxanthin in animal body - Google Patents
Method for testing residual canthaxanthin in animal body Download PDFInfo
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Abstract
The invention relates to a method for testing residual canthaxanthin in an animal body, belonging to the technical field of food safety testing. The testing method is an improvement based on the prior art. The improvement includes: a, preparing standard canthaxanthin solution; weighing a certain amount of standard canthaxanthin, placing the canthaxanthin in a 100ml brown volumetric flask, adding N and N-dimethylformamide, fixing the volume to be 100ml, shaking, and storing the solution in a refrigerator at minus 4 DEG C as stock solution; respectively weighing a certain amount of stock solution, and respectively preparing 0.05mu g/ml standard solution, 0.1mu g/ml standard solution, 0.2mu g/ml standard solution, 0.5mu g/ml standard solution, 1.0mu g/ml standard solution, 2.0mu g/ml standard solution, 5.0mu g/ml standard solution, 10.0mu g/ml standard solution and 20.0mu g/ml standard solution with methanol solution; and b, chromatographic conditions: using a Waters2695 high-performance liquid chromatograph and an ultraviolet detector, with the chromatographic column being Symmetry C18 5mu m, 3.9 * 150mm, the wavelength being 475nm, the flow rate being 1.0ml/min, the mobile phase adopting acetonitrile and water with the volume ratio being 95: 5, the column temperature being 30 DEG C, and the sample injection volume being 50mu l. The invention has the advantages of simple and rapid operation and accurate and reliable results, and the method is suitable for analysis and detection of large-volume samples.
Description
Technical field:
The present invention relates to the residual detection method of canthaxanthin in a kind of animal body, belong to the food safety detection technical field.
Background technology:
In recent years, along with improving constantly of China's living standards of the people, people strengthen year by year to the consumption demand of animal derived food, but the animal derived food safety problem is very outstanding, except various infecting both domestic animals and human diseases such as rabid ox disease, aftosa, bird flu threaten, great food security incidents such as clenbuterol hydrochloride, chloromycetin, tonyred, melamine also frequently take place, and people are more and more paid close attention to the problem of abuse adjuvant in the food.Particularly add tonyred and caused since " red-yolk duck egg " disturbance from food, the abuse of feeding colorant and harm have caused people's extensive concern.
Canthaxanthin (Canthaxanthin) claim canthaxanthin, cantharides xanthin again, be a kind of orange-red carotenoid of being with, natural being present in many foods, for example mushroom, shellfish, fish and eggs, it can be produced by microbe culture or chemosynthesis.Canthaxanthin can change the color and luster of poultry and yolk mainly as the animal feed colorant, be widely used with bird feed in, coverage rate reaches 90%.This colorant without any help, and is eaten the colour of skin that such chicken and egg might have influence on the people for a long time aspect the value that has additional nutrients except the color that changes chicken body and egg.The Food Science council of European Union also once found to add the red principal ingredient canthaxanthin absorption of beautiful element and got in touch with the existence of human body PVR, and the high intake of canthaxanthin may cause pigment in amphiblestroid accumulation, thereby affects one's power of vision.Therefore for safer, reasonably use feeding colorant, ensure animal derived food safety, canthaxanthin is carried out the residue detection analysis in animal body, very great and profound significance are arranged.
At present, less about the method for detecting residue of canthaxanthin in liver, only a small amount of detection method about canthaxanthin, mainly contain spectrophotometer method, this method mainly is to detect the addition of canthaxanthin in the feed, for the residual condition of canthaxanthin in organizing internal organs, lacks certain feasibility.Spectrophotometer method can only be at the many situations of residual quantity, and the result is undesirable after a little while for measuring.Also relevant for the research with canthaxanthin in the high effective liquid chromatography for measuring feed, canthaxanthin has formal and trans two kinds of structures in addition, and this method can only be isolated a kind of structure, and is used for extracting when organizing internal organs, and the recovery is lower, can not satisfy experimental requirements.Therefore, method for detecting residue is significant for food security that promotes China and guarantee people health fast and accurately in animal body to set up canthaxanthin.
Summary of the invention:
The object of the present invention is to provide a kind ofly simple to be suitable for the analyzing and testing of batch samples fast, the result accurately, the residual detection method of canthaxanthin in the animal body reliably.
The present invention adopts the residual research of canthaxanthin in the high effective liquid chromatography for measuring animal body inner tissue internal organs, this method is by the research to canthaxanthin standard compound method, and the selection of chromatographic condition, canthaxanthin is separated from tissue extract completely, and energy success the formal structure of canthaxanthin is separated fully with transconfiguration, thereby the content of accurately qualitative, quantitative assay determination canthaxanthin.This method is simply quick, is suitable for the analyzing and testing of batch samples, and the result accurately, reliably.
The residual detection method of canthaxanthin is the improvement on the prior art basis in the animal body of the present invention, and its improvement is:
1, the preparation of canthaxanthin standard solution
Take by weighing a certain amount of canthaxanthin standard items, place the brown volumetric flask of 100ml, add N, dinethylformamide is settled to 100ml, shakes up, and is stored in-4 ℃ of refrigerators as storing solution.Take by weighing a certain amount of storing solution respectively, be mixed with the standard solution of variable concentrations with methanol solution respectively: 0.05 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.5 μ g/ml, 1.0 μ g/ml, 2.0 μ g/ml, 5.0 μ g/ml, 10.0 μ g/ml and 20.0 μ g/ml.
2, chromatographic condition: the attached UV-detector of Waters2695 high performance liquid chromatograph, chromatographic column Symmetry C185 μ m, 3.9 * 150mm, wavelength 475nm, flow velocity 1.0ml/min, moving phase V (acetonitrile): V (water)=95: 5,30 ℃ of column temperatures, sample size 50 μ l.
The present invention compared with prior art has that this is simply quick, the result accurately, advantage reliably, be suitable for the analyzing and testing of batch samples.
Description of drawings:
Fig. 1 is the chromatogram (concentration is 0.5 μ g/ml) after with acetonitrile storing solution being diluted.
Fig. 2 is the chromatogram (concentration is 0.5 μ g/ml) after with methyl alcohol storing solution being diluted.
Fig. 3 is the wavelength with ultraviolet spectrophotometer scan angle flavine.
Fig. 4 is the chromatogram (being followed successively by from left to right 100: 0,95: 5,90: 10,85: 15) of moving phase acetonitrile and water different proportion.
Fig. 5 is the chromatogram (being followed successively by from left to right 100: 0,95: 5,90: 10) of mobile phase methanol and water different proportion.
Fig. 6 is the asynchronous chromatogram of column temperature (being followed successively by 55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃, 30 ℃, 25 ℃, 20 ℃ from left to right).
Fig. 7 is the asynchronous chromatogram of flow velocity (being followed successively by 1.2ml/min, 1.0ml/min, 0.7ml/min from left to right).
Fig. 8 is a canthaxanthin repeated experiment chromatogram.
Fig. 9 is the canthaxanthin typical curve.
Figure 10 is a canthaxanthin stability experiment chromatogram.
Embodiment:
The residual detection method of canthaxanthin is the improvement on the prior art basis in the animal body of the present invention, and its concrete steps are as follows:
1, the preparation of canthaxanthin standard solution
(1) takes by weighing a certain amount of canthaxanthin standard items, place the brown volumetric flask of 100ml.
(2) 1. add N, dinethylformamide is settled to 100ml, shakes up, and is stored in-4 ℃ of refrigerators as storing solution.
2. add acetonitrile and be settled to 100ml, shake up, as storing solution preserve with-4 ℃ of refrigerators in.
By observing, use N, dinethylformamide dissolving canthaxanthin can make it dissolve fully, and the solution clear, and color is darker; With acetonitrile dissolving canthaxanthin it is dissolved fully, have particle to exist, and the solution muddiness, color is more shallow.So we select N for use in this step, dinethylformamide dissolving canthaxanthin also is settled to 100ml, is prepared into storing solution.
(3) take by weighing a certain amount of storing solution respectively, be mixed with the standard solution of variable concentrations: 0.05 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.5 μ g/ml, 1.0 μ g/ml, 2.0 μ g/ml, 5.0 μ g/ml, 10.0 μ g/ml and 20.0 μ g/ml.
1. with acetonitrile storing solution is diluted to the standard solution of above variable concentrations, uses high effective liquid chromatography for measuring;
2. with methyl alcohol storing solution is diluted to the standard solution of above variable concentrations, uses high effective liquid chromatography for measuring;
Find out that by Fig. 1, Fig. 2 the peak type that the standard solution that disposes with acetonitrile occurs is bad, unstability of base line, peak area is less; It is relatively good the peak type that each concentration occurs to occur with the standard solution of methyl alcohol configuration, baseline stability, and peak area is normal.We select the methyl alcohol preparing standard solution for use so in experiment from now on.
2, the pre-treatment of sample
Tissue accurately takes by weighing being organized in the 50ml centrifuge tube that 3g pulverized after thawing, add 30ml acetonitrile and 5g anhydrous sodium sulfate, vortex mixing 1min, vibration 10min, ultrasonic 1min, the centrifugal 5min of 3000r/min then moves into supernatant in the separating funnel of the 125ml that presets the 20ml normal hexane (separating funnel is hunted leak with acetonitrile with preceding need) standing demix behind the mixing; Collect lower floor's acetonitrile phase, normal hexane is stayed in the separating funnel mutually.
By above-mentioned steps residue in the centrifuge tube is repeated to extract 2 times with 20ml acetonitrile extraction agent more at every turn, merge collected acetonitrile phase after three degreasings, add 5ml n-propanol (reduction boiling point), be concentrated into dried in rotary evaporation below 40 ℃.
Accurately draw 5ml methyl alcohol, in the rotary evaporation bottle, ultrasonic 1min, fully the mixing content places and measures in the bottle.Directly going up high performance liquid chromatograph behind 0.45 μ m filtering with microporous membrane measures.
3, the selection of wavelength
Found out that by Fig. 3 the sample liquid that will contain canthaxanthin carries out length scanning by ultraviolet-visible pectrophotometer, at wavelength 475nm place absorption maximum is arranged, the preferred detection wavelength is 475nm.
4, the selection of moving phase
Separate the used moving phase of carotenoid material and be mostly the solvent of selecting for use polarity stronger, as acetonitrile, methyl alcohol, normal hexane etc.Therefore we select acetonitrile, the methyl alcohol alternative as moving phase for use, and on column temperature was 30 ℃, the basis of flow velocity 1.0ml/min, the adjustment by ratio between them and between the water finally obtained our required ratio.
Find out by Fig. 4, Fig. 5, the selection of ratio between acetonitrile and the water: acetonitrile was made as respectively 100: 0,95: 5,90: 10,85: 15 than water, the result finds out by liquid phase, the ratio of acetonitrile and water is 95: 5 o'clock, canthaxanthin formally thoroughly can not only be separated with trans, also have good peak type, retention time is short.The selection of ratio between methyl alcohol and the water: methyl alcohol was made as respectively 100: 0,95: 5,90: 10 than water, the result finds out by liquid phase, and the ratio of methyl alcohol and water is 95: 5 o'clock, canthaxanthin formally thoroughly can not only be separated with trans, also have good peak type, retention time is less.
By above two kinds of comparisons, we select acetonitrile than water 95: 5 and methyl alcohol than water 95: 5, they can both obtain good result, but acetonitrile and water mixing energy form the polar solvent of high-k, canthaxanthin can better be separated.Therefore select acetonitrile than water 95: 5 as moving phase.
5, the selection of column temperature
In moving phase is on the basis of acetonitrile than water 95: 5, flow velocity 1.0ml/min, adjusts the temperature of chromatographic column, finally selects a suitable temperature.Column temperature is set at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ respectively, find out by Fig. 6, rising along with temperature, retention time is shorter and shorter, because canthaxanthin is heated with easy decomposition, consider that again the material organized in the internal organs is complicated, the assorted peak of front is many, through relatively we to select column temperature be 30 ℃.
6, the selection of flow velocity
In moving phase is that acetonitrile is on 30 ℃ the basis than water 95: 5, column temperature, by adjusting flow velocity, finally selects a suitable flow velocity.Flow velocity is set at 0.7ml/min, 1.0ml/min, 1.2ml/min respectively, is found out by Fig. 7, along with the rising of flow velocity, retention time is shorter and shorter.When flow velocity was 1.0ml/min, it is better that retention time suits to cut the peak type, is 1.0ml/min so we select the flow velocity of moving phase.
7, repeatability, the range of linearity, regression equation and detection limit
Draw reference substance solution 50L, replication 6 times, canthaxanthin peak retention time fluctuates less than 0.1min (the results are shown in Figure 8).The relative standard deviation RSD of peak area is 0.43%, shows that method precision is good.The standard solution of preparation series concentration is measured, and is ordinate with the peak area y of canthaxanthin, and the mass concentration x of canthaxanthin (μ g/ml) is a horizontal ordinate drawing standard curve, is found out by Fig. 9, and regression equation is y=576685x-35679, related coefficient (R
2) be 0.9994 (n=9), show that canthaxanthin is good in mass concentration 0.05-20.0 μ g/ml scope internal linear relation.Determine that with 3 times of signal to noise ratio (S/N ratio)s (S/N) detecting of this method is limited to 0.06mg/kg.
8, recovery experiment
Accurately take by weighing sample 5.0g, add canthaxanthin content and be respectively 0.1,0.2,0.5,1.0 and 5 levels of 2.0mg/kg, do recovery experiment, measure canthaxanthin content by above chromatographic condition, each sample introduction 50 μ l.Found out that by table 1 recovery is 88.9-98.1%, RSD is 0.93-3.34%, and recovery result is good.
Table 1 canthaxanthin determination of recovery rates result (n=3)
Table?1?Recovery?results?of?eanthaxanthin(n，＝3)
9, stability experiment
Get the canthaxanthin standard solution 50ul sample introduction of the 5.0mg/L for preparing, measure (Figure 10 as a result) every a period of time.Peak area relative standard deviation (RSD) is 0.78%, and trans and formal structure retention time is respectively 0.15% and 0.17%, shows that this stability of sample is better.
10, the mensuration of sample
The same even sample of saying by the front of sample-pretreating method re-treatment in the daytime 6 times calculates canthaxanthin content ug/g with external standard method, and the canthaxanthin average content is 0.118g in the sample, and relative standard deviation (RSD) is 0.79%, the results are shown in Table 2.
The content of canthaxanthin in table 2 sample
Table?2?The?Content?of?Eanthaxanthin?in?Samples(n，＝3)
Claims (1)
1. the interior residual detection method of canthaxanthin of animal body is the improvement on the prior art basis, and its improvement is:
A. the preparation of canthaxanthin standard solution: take by weighing a certain amount of canthaxanthin standard items, place the brown volumetric flask of 100ml, add N, dinethylformamide is settled to 100ml, shakes up, and is stored in-4 ℃ of refrigerators as storing solution; Take by weighing a certain amount of storing solution respectively, be mixed with the standard solution of variable concentrations with methanol solution respectively: 0.05 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.5 μ g/ml, 1.0 μ g/ml, 2.0 μ g/ml, 5.0 μ g/ml, 10.0 μ g/ml and 20.0 μ g/ml;
B. chromatographic condition: the attached UV-detector of Waters2695 high performance liquid chromatograph, chromatographic column Symmetry C185 μ m, 3.9 * 150mm, wavelength 475nm, flow velocity 1.0ml/min, moving phase V (acetonitrile): V (water)=95: 5,30 ℃ of column temperatures, sample size 50 μ l.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110672769A (en) * | 2019-10-27 | 2020-01-10 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring residual quantity of cantharidin yellow pigment in poultry eggs |
| CN110749689A (en) * | 2019-11-07 | 2020-02-04 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring content of cantharis yellow colorant in feed |
| CN112379024A (en) * | 2020-12-08 | 2021-02-19 | 浙江大学 | Solid phase extraction-high performance liquid chromatography for determining cantharis yellow in egg yolk |
| CN113009045A (en) * | 2021-03-29 | 2021-06-22 | 浙江大学 | Method for measuring cantharis yellow in feed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110672769A (en) * | 2019-10-27 | 2020-01-10 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring residual quantity of cantharidin yellow pigment in poultry eggs |
| CN110749689A (en) * | 2019-11-07 | 2020-02-04 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring content of cantharis yellow colorant in feed |
| CN112379024A (en) * | 2020-12-08 | 2021-02-19 | 浙江大学 | Solid phase extraction-high performance liquid chromatography for determining cantharis yellow in egg yolk |
| CN112379024B (en) * | 2020-12-08 | 2021-12-07 | 浙江大学 | Solid phase extraction-high performance liquid chromatography for determining cantharis yellow in egg yolk |
| CN113009045A (en) * | 2021-03-29 | 2021-06-22 | 浙江大学 | Method for measuring cantharis yellow in feed |
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Application publication date: 20111221 |