CN114910580B - Detection method of beta-apo-8' -ethyl carotenate - Google Patents

Detection method of beta-apo-8' -ethyl carotenate Download PDF

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CN114910580B
CN114910580B CN202210392961.6A CN202210392961A CN114910580B CN 114910580 B CN114910580 B CN 114910580B CN 202210392961 A CN202210392961 A CN 202210392961A CN 114910580 B CN114910580 B CN 114910580B
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beta
ethyl
carotene
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CN114910580A (en
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何开蓉
梁玉树
乔煦玮
熊安琪
刘尚群
王力
刘珊
陈霞
吴雨聪
马金凤
周桂莲
李勇
孔凡科
郭丽君
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CHENGDU CENTURY INVESTMENT CO LTD
Sichuan New Hope Liuhe Technology Innovation Co ltd
Weifang New Hope Liuhe Feed Technology Co ltd
Shandong New Hope Liuhe Group Co Ltd
New Hope Liuhe Co Ltd
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CHENGDU CENTURY INVESTMENT CO LTD
Sichuan New Hope Liuhe Technology Innovation Co ltd
Weifang New Hope Liuhe Feed Technology Co ltd
Shandong New Hope Liuhe Group Co Ltd
New Hope Liuhe Co Ltd
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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
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
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Abstract

The invention discloses a detection method of beta-apo-8' -ethyl carotene, which comprises the following steps: (1) preparing a sample solution to be tested and a standard working solution; (2) Detecting a sample solution to be detected and a standard working solution by using a high performance liquid chromatograph, and recording a chromatogram; (3) Obtaining peak areas according to chromatograms of standard working solutions with different concentrations, and performing linear regression analysis to obtain a standard curve linear regression equation; and analyzing a chromatogram of the sample solution to be detected to obtain a peak area, substituting the peak area into a standard curve linear regression equation to obtain the mass concentration of the beta-apo-8 '-ethyl carotenoate in the sample solution to be detected, and calculating to obtain the content of the beta-apo-8' -ethyl carotenoate in the feed or the additive. The method disclosed by the invention is simple in pretreatment operation, high in accuracy and good in reproducibility, the sensitivity and the precision of the method can meet the requirements of practical application, and the method is suitable for batch detection of the content of the aporate in the feed.

Description

Detection method of beta-apo-8' -ethyl carotenate
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a detection method of beta-apo-8' -ethyl carotene.
Background
The beta-apo-8' -ethyl carotene is abbreviated as apo-ester, is an oxygen-containing apo-carotene compound in carotenoid, is mainly a metabolite of apo-carotene aldehyde in green grass, alfalfa, green vegetables and citrus fruits, has a structural formula shown in figure 1, has a molecular formula of C 32H44O2, has a molecular weight of 460.7, is easily dissolved in chloroform, is slightly dissolved in vegetable oil, is extremely insoluble in water and glycerol, and is extremely unstable to oxygen and light. The aporate is a high-efficiency carotenoid additive, the coloring efficiency of the aporate is obviously higher than that of beta-apo-8 '-carotenal and marigold extract, the aporate is commonly applied to the feed industry, the aporate has obvious effect on coloring skin, foot shin, subcutaneous fat and egg yolk of poultry, and the total amount of the aporate is not more than 80mg/kg when the aporate is matched with capsanthin, beta-apo-8' -carotenal, canthaxanthin or lutein for use.
The national standard for detecting the beta-apo-8 '-ethyl carotene is mainly spectrophotometry, and the common detection method in the practical application process is ultra-high performance liquid chromatography, which adopts ultra-high performance liquid chromatography-tandem mass spectrometry, but the ultra-high performance liquid chromatography has high detection cost and complex flow, so that the research on the detection method of the beta-apo-8' -ethyl carotene is urgently needed.
Disclosure of Invention
The invention aims to solve the problems of high cost and complex flow of an ultra-high performance liquid chromatography in the prior art and provides a detection method of beta-apo-8' -ethyl carotene, which adopts simple sample pretreatment and high performance liquid chromatography to rapidly and accurately determine the content of apo-ester in feed or additive, thereby effectively helping to monitor and evaluate the use of coloring agent in feed and ensuring the quality safety of feed.
In order to achieve the above object, the present invention provides the following technical solutions:
a detection method of beta-apo-8' -ethyl carotene comprises the following steps:
(1) Preparation of sample solution to be tested and standard working solution
Weighing X a g of feed or X b g of additive into a 100mL volumetric flask, adding 2-10mL of water, and uniformly mixing by vortex; adding 15-30mL of methyl tertiary butyl ether, and uniformly mixing by shaking; adding methanol, and shaking and mixing uniformly; methanol is used for fixing the volume to 100mL, and the mixture is shaken uniformly and then is stood to obtain a sample solution to be measured;
The volume ratio is 15-30: preparing 70-85 methyl tertiary butyl ether-methanol mixed solution into beta-apo-8' -ethyl carotin standard working solution with different concentrations;
(2) High performance liquid chromatography
Detecting the sample solution to be detected and the standard working solution obtained in the step (1) by using a high performance liquid chromatograph, and recording a chromatogram;
(3) Standard curve drawing and beta-apo-8' -ethyl carotin content calculation
Obtaining peak areas according to chromatograms of beta-apo-8' -ethyl carotenoate standard working solutions with different concentrations, and carrying out linear regression analysis on the peak areas and the corresponding mass concentrations to obtain a standard curve linear regression equation;
Analyzing a chromatogram of a sample solution to be detected to obtain a peak area, substituting a standard curve linear regression equation to obtain the mass concentration of beta-apo-8 '-ethyl carotenoate in the sample solution to be detected, and then calculating to obtain the content of beta-apo-8' -ethyl carotenoate in the feed or additive; the calculation formula of the beta-apo-8' -ethyl carotenate is as follows:
wherein W is the content of beta-apo-8 '-ethyl carotene in the feed or the additive, x is the mass concentration of beta-apo-8' -ethyl carotene in the sample solution to be detected, V is the volume of the solution used for preparing the sample solution to be detected, and m is the mass of the feed or the additive.
Further, in the step (1), the sample solution to be tested of the feed sample is concentrated, specifically: taking 5ml of supernatant solution from a 100ml volumetric flask into a centrifuge tube, blow-drying by a nitrogen blowing instrument, and transferring 1ml of supernatant solution into a centrifuge tube with a volume ratio of 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution is re-dissolved and filtered to obtain a sample solution to be tested of a feed sample.
Further, the mass of the feed was weighed to be 2.0g.
Further, in the step (1), the sample solution to be tested of the additive sample is diluted, and the specific operation is as follows: taking 5mL of supernatant solution from a 100mL volumetric flask into the 100mL volumetric flask, and using the volume ratio of 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution to a fixed volume, and filtering to obtain a sample solution to be tested of the additive sample.
Further, the mass of the additive was weighed to be 0.05g.
Further, the conditions of the high performance liquid chromatograph are: adopting a C30 column, wherein the detection wavelength is 466nm, the column temperature is 30-35 ℃, and the mobile phase is 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution; the flow rate of the elution is 0.8-1.5 ml/min. C30 The column is a reversed phase chromatographic column with C30 functional groups bonded on a silica gel matrix, the hydrophobicity of a stationary phase is increased, and the product is very effective for separating geometric isomers such as carotenoid and the like. The method adopts C30 chromatographic column to separate lutein, zeaxanthin, canthaxanthin, beta-apo-8' -ethyl carotene, etc.
Further, the column temperature was 35 ℃, and the mobile phase was 15% by volume: 85 methyl tert-butyl ether-methanol mixed solution; the flow rate of elution is 1.0ml/min, and the sample injection amount is 50 mu L; run time was 20min.
Further, the standard curve linear regression equation is: y= 479.074865x-1.4006713, y is the peak area of the beta-apo-8 '-ethyl carotene, x is the mass concentration of the beta-apo-8' -ethyl carotene in the sample solution to be detected, and the correlation coefficient R2=1.00000.
Further, in the step (1), the feed mass concentration of the feed sample solution to be tested is 100mg/mL, and the additive mass concentration of the additive sample solution to be tested is 0.025mg/mL.
Further, the lower limit of detection of the content of beta-apo-8' -ethyl carotene in the feed is 0.15mg/kg.
Further, the quantitative detection concentration of the content of the beta-apo-8' -ethyl carotene in the feed is 0.4mg/kg.
Compared with the prior art, the invention has the beneficial effects that:
The invention discloses a method for detecting beta-apo-8 '-ethyl carotene, which adopts the pretreatment of water dissolution and extraction and then methyl tertiary butyl ether-methanol mixed solution extraction, has good selectivity to apo ester, has little impurity content and less interference of solution to be detected, and can be used for measuring the content of beta-apo-8' -ethyl carotene in feed or additive by matching with conventional high performance liquid chromatography analysis. Experiments prove that the detection limit of the method is 0.15mg/kg, the quantitative limit is 0.40mg/kg, all technical indexes meet the requirements, and the content of lutein, zeaxanthin or canthaxanthin in the feed or the additive can be detected. Meanwhile, the pretreatment method is simple in operation, high in accuracy and good in reproducibility, the sensitivity and the precision of the method can meet the requirements of practical application, the method is suitable for batch detection of the content of the aporate in the feed, and the method has a certain reference value for perfecting the chromatographic detection method in the feed.
Description of the drawings:
FIG. 1 is a structural formula of beta-apo-8' -carotin acid ethyl ester;
FIG. 2 is a chromatogram of ethyl beta-apo-8' -carotene using n-hexane as the extract;
FIG. 3 is a chromatogram of ethyl beta-apo-8' -carotene using methylene chloride as the extract;
FIG. 4 is a chromatogram of ethyl beta-apo-8' -carotene using cyclohexane as the extract;
FIG. 5 is a chromatogram of ethyl beta-apo-8' -carotene using methanol as the extract;
FIG. 6 is a chromatogram of beta-apo-8' -carotin acid ethyl ester as extract with a volume ratio of 30:70 methyl tert-butyl ether-methanol mixed solution;
FIG. 7 is a standard curve linear regression equation;
FIG. 8 is a chromatogram for separating lutein, zeaxanthin, canthaxanthin, and beta-apo-8' -ethyl carotene from layer feed 1 #;
FIG. 9 is a blank labeled recovery chromatogram of a feed sample;
FIG. 10 is a chromatogram of feed 0.4mg/kg for standard recovery.
Detailed Description
The present invention will be described in further detail with reference to test examples and specific embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following embodiments, and all techniques realized based on the present invention are within the scope of the present invention.
Example 1
A. Instrument and reagent
High performance liquid chromatograph (Agilent 1260 index II); an ultraviolet detector (1260 MWD); centrifuge (Althaeae); a fully automatic concentrator (ReekoAutoEVA); HY-6 double-layer speed regulating oscillator; microporous organic filters (0.22 μm, shanghai' an Pu); vortex meter.
Beta-apo-8' -ethyl carotene standard (purity is more than or equal to 98.6%, carote Nature); methyl tertiary butyl ether (chromatographic purity), methanol (chromatographic purity); chloroform, n-hexane, cyclohexane (analytically pure).
B. Determination of mixing mode
In the production of beta-apo-8' -carotin acid ethyl ester, water-soluble matrix coating is adopted, in order to open the coating layer of apo-ester in the feed, firstly, a sample is placed in a proper amount of water for dissolution, and then, an extracting solution is used for extraction. When extracting by using the extracting solution, mixing is carried out in two modes of oscillation and ultrasound respectively, and experiments show that the solution temperature is easy to rise by adopting the ultrasound, and the mixing time cannot be set longer, so that the embodiment adopts the mixing mode of oscillation.
C. determination of the extract
In the embodiment, 2.0g of a blank feed sample is weighed, a specified amount of beta-apo-8 '-ethyl carotenoate standard solution is added into the feed blank sample, n-hexane, chloroform, cyclohexane, methyl tert-butyl ether-methanol mixed solution, methanol and methanol-ethyl acetate-n-hexane mixed solution are respectively adopted as extracting solutions, the content of beta-apo-8' -ethyl carotenoate is detected after the sample is extracted, the recovery rate is calculated, and 3 groups of parallel samples are prepared from each extracting solution in the test process. FIGS. 2 to 6 are chromatograms of ethyl beta-apo-8 '-carotenoate using n-hexane, chloroform, cyclohexane, methyl tert-butyl ether and methanol mixed solution, methanol, ethyl acetate and n-hexane mixed solution as the extract, respectively, and the recovery rates of ethyl beta-apo-8' -carotenoate are shown in Table 1. The result shows that the extraction interference of the chloroform and the methyl tertiary butyl ether-methanol is less, and the recovery rate is high. But chloroform is a toxic and harmful water pollutant and belongs to a second class of easily-made reagents, and is not easy to obtain, so that methyl tertiary butyl ether-methanol is selected as an extracting solution. Meanwhile, methyl tertiary butyl ether-methanol extraction solvents with different proportions are optimized, table 1 shows the recovery rate of 10% -70% of methyl tertiary butyl ether in the methyl tertiary butyl ether-methanol mixed solution in the extraction solution, and from the data, it can be seen that when the methyl tertiary butyl ether proportion in the methyl tertiary butyl ether-methanol mixed solution is not less than 15%, the recovery rate is more than 90%, the test requirement of beta-apo-8' -ethyl carotene is met, and table 2 shows the peak areas of lutein and zeaxanthin tested by taking the methyl tertiary butyl ether-methanol mixed solution with 15% -70% of methyl tertiary butyl ether as the extraction solution, the extraction efficiency of lutein and zeaxanthin gradually decreases with the increase of the methyl tertiary butyl ether proportion. Considering the characteristics of high volatility, high toxicity, difficult mutual solubility with water and the like of the methyl tertiary butyl ether, the proportion of the methyl tertiary butyl ether is not too high from the safety aspect, so the volume ratio is selected to be 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution is used as an extracting solution.
TABLE 1 recovery of beta-apo-8' -carotin acid ethyl ester with different extracts
TABLE 2 extraction efficiency of other pigments with different ratio methyl tert-butyl ether-methanol mixed solution
D. Ultraviolet absorption wavelength determination
The maximum ultraviolet absorption wavelength of the beta-apo-8' -ethyl carotene in normal hexane is 466nm, and according to other data, the wavelength is measured by adopting 466, 452 and 472nm to 2.0mg/L apo-ester standard solution, and the result shows that the detection peak area is not changed obviously, and in the embodiment, 466nm is selected as the detection wavelength.
E. chromatographic column determination
The C30 column is a reversed phase chromatographic column bonded with a C30 functional group on a silica gel matrix, and the hydrophobicity of a stationary phase is added, so that the product is very effective for separating geometric isomers such as carotenoid and the like. The invention adopts the C30 chromatographic column to separate the beta-apo-8' -ethyl carotene.
A detection method of beta-apo-8' -ethyl carotene comprises the following steps:
(1) Preparation of sample solution to be tested and standard working solution
Weighing 2.0 feed or 0.05g additive into a 100mL volumetric flask, adding 2-10mL water, and mixing by vortex for 2min; adding 30mL of methyl tertiary butyl ether, and shaking and uniformly mixing for 10min; adding methanol, shaking by a shaking table, and mixing for 30min; and (3) using methanol to fix the volume to 100mL, shaking uniformly, and standing to obtain a sample solution to be detected.
For the sample solution to be tested of the feed sample, concentration is also required, and the specific operation is as follows: taking 5ml of supernatant solution from a 100ml volumetric flask into a centrifuge tube, drying the supernatant solution at 45 ℃ by a nitrogen blower, and transferring 1ml of supernatant solution into a centrifuge tube with a volume ratio of 30:70, re-dissolving the methyl tertiary butyl ether-methanol mixed solution, and filtering to obtain a sample solution to be tested of the feed sample. The filtering is carried out by adopting an organic filter membrane with the thickness of 0.22 mu m, and impurities are not filtered, so that chromatographic column blockage is easy to cause, and detection is influenced.
The method comprises the following specific operations of diluting a sample solution to be tested of an additive sample: 5mL of the supernatant solution was taken from a 100mL volumetric flask in a 100mL volumetric flask with a volume ratio of 30:70, the mixed solution of methyl tertiary butyl ether and methanol is subjected to constant volume and filtration to obtain a sample solution to be tested of the additive sample.
Completely transferring 1.0mg of beta-apo-8' -carotin acid ethyl ester into a 25mL volumetric flask, adding 7mL of methyl tertiary butyl ether for dissolution, and using methanol to make volume to 25mL, thus obtaining 40 mug/mL apo ester standard solution, and preserving at-18 ℃. Different volumes of 40 mug/mL of aporate standard solution are removed, and the volume ratio is 30:70, and respectively preparing 0.04, 0.1, 0.2, 1.0, 2.0 and 10.0 mug/mL of aporate standard solution.
The solution preparation process of this example was carried out in a fume hood protected from light.
(2) High performance liquid chromatography
And (3) detecting the sample solution to be detected obtained in the step (1) and standard working solutions with different concentrations by using a high performance liquid chromatograph, and recording a chromatogram.
The conditions of the high performance liquid chromatograph are as follows: the detection wavelength was 466nm using a C30 column (250X 4.6mml. D.S-5 μm), the column temperature was 35℃and the mobile phase was 15% by volume: 85 methyl tertiary butyl ether and methanol mixed solution, the eluting flow rate is 1.0ml/min, and the sample injection amount is 50 mu L; run time was 20min.
(3) Standard curve drawing and beta-apo-8' -ethyl carotin content calculation
And (3) analyzing the chromatograms of the standard working solutions of the beta-apo-8' -ethyl carotenoate with different concentrations to obtain peak areas, performing linear regression analysis on the peak areas and the corresponding mass concentrations, wherein the apo-ester concentration is on the abscissa, and the peak areas are on the ordinate, so as to obtain a standard curve linear regression equation. The result shows that in the concentration range of 0.04-10 mg/L, the chromatographic peak area of the aporate and the concentration are in good linear relation, and the standard curve linear regression equation is as follows: y= 479.074865x-1.4006713, y is the peak area of the beta-apo-8 '-ethyl carotene, x is the mass concentration of the beta-apo-8' -ethyl carotene in the sample solution to be detected, and the correlation coefficient R 2 =1.00000, and the standard curve is shown in fig. 7.
Analyzing a chromatogram of a sample solution to be detected to obtain a peak area, substituting a standard curve linear regression equation to obtain the mass concentration of beta-apo-8 '-ethyl carotenoate in the sample solution to be detected, and then calculating to obtain the content of beta-apo-8' -ethyl carotenoate in the feed or additive; the calculation formula of the beta-apo-8' -ethyl carotenate is as follows:
Wherein W is the content of beta-apo-8 '-ethyl carotene in the feed or additive, x is the mass concentration of beta-apo-8' -ethyl carotene in the sample solution to be tested, V is the volume of the solution used for preparing the sample solution to be tested, and m is the mass of the feed or additive, in this embodiment, after the feed sample is prepared into 100ml of solution, 5ml is taken and concentrated into 1ml, then For the additive sample, after preparing 100ml of solution, 5ml of the solution is diluted to 100ml, then
The contents of beta-apo-8' -ethyl carotenate obtained by the detection of different feed samples and different additive samples by the method described above are shown in the following table 3.
TABLE 3 content of beta-apo-8' -caroteneic acid ethyl ester in different feed samples and different additives
From the data in Table 2 above, it can be seen that the amount of aporate in the 10% aporate additive was detected to be substantially 10% by the method of the present invention. For the feed samples, the content of apoester, lutein, zeaxanthin and canthaxanthin can be detected, wherein canthaxanthin is not added in the 510-meat chickens. Wherein FIG. 8 is a chromatogram of separating lutein, zeaxanthin, canthaxanthin, and beta-apo-8' -ethyl carotene from layer feed 1 #. The poultry cannot synthesize pigment per se, and only carotenoids with oxygen-containing functional groups comprise lutein, lycopene, canthaxanthin, aporate and the like. Therefore, the research on the coloring effect of the lutein coloring agent on the yellow chickens and the coloring and lutein depositing effects on the egg yolk of the laying hens has important guiding significance for guiding production practice and improving the quality of products.
In order to test the accuracy and precision of the method, 2.0g of a blank feed sample is accurately weighed, a proper amount of beta-apo-8' -ethyl carotenoate standard solution is added into the blank feed sample, the addition amount of apo-ester in a sample is respectively made to be 0.4, 1.0 and 10.0mg/kg 3 levels, 6 repeated samples are made for each level, the recovery rate and RSD are calculated according to the detection amount and theoretical addition amount, and the accuracy and precision of the method are inspected. The test data are shown in the following table 4, and it is found that the recovery rates of aporate are 98.5%, 99.8%, 86.0%, and RSD are 2.87%, 4.73%, and 4.66%, respectively, which indicate that the accuracy and precision of the method are good.
TABLE 4 recovery of aporate with different addition levels
Additive amount (mg/kg) Average recovery (%) RSD(％)
0.4 98.5 2.87
1.0 99.8 4.73
10 86.0 4.66
In order to test the detection limit and the quantitative limit of the method, blank samples are taken as matrixes and marked, the concentration of the apoate in the samples corresponding to the signal-to-noise ratio (S/N=3) is taken as the detection limit, and the concentration of the apoate in the samples corresponding to the signal-to-noise ratio (S/N=10) is taken as the quantitative limit, so that the detection limit is 0.15mg/kg; the limit of quantification was 0.4mg/kg. Sample blank and 0.4mg/kg are recovered by labeling as shown in FIG. 9 and FIG. 10.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (10)

1. The detection method of the beta-apo-8' -ethyl carotene is characterized by comprising the following steps of:
(1) Preparation of sample solution to be tested and standard working solution
Weighing X a g of feed or X b g of additive into a 100 mL volumetric flask, adding 2-10mL of water, and uniformly mixing by vortex; adding 15-30mL of methyl tertiary butyl ether, and uniformly mixing by shaking; adding methanol, and shaking and mixing uniformly; methanol is used for fixing the volume to 100 mL, and the mixture is shaken uniformly and then is stood to obtain a sample solution to be measured;
the volume ratio is 15-30: preparing 70-85 methyl tertiary butyl ether-methanol mixed solution into beta-apo-8' -ethyl carotin standard working solution with different concentrations;
(2) High performance liquid chromatography
Detecting the sample solution to be detected and the standard working solution obtained in the step (1) by using a high performance liquid chromatograph, and recording a chromatogram;
(3) Standard curve drawing and beta-apo-8' -ethyl carotene content calculation
Obtaining peak areas according to the chromatograms of beta-apo-8' -ethyl carotene standard working solutions with different concentrations, and carrying out linear regression analysis on the peak areas and the corresponding mass concentrations to obtain a standard curve linear regression equation;
Analyzing a chromatogram of a sample solution to be detected to obtain a peak area, substituting the peak area into a standard curve linear regression equation to obtain the mass concentration of beta-apo-8 '-ethyl carotenoate in the sample solution to be detected, and then calculating to obtain the content of beta-apo-8' -ethyl carotenoate in the feed or additive; the calculation formula of the beta-apo-8' -carotin acid ethyl ester is as follows:
Wherein W is the content of beta-apo-8 '-ethyl carotene in the feed or additive, x is the mass concentration of beta-apo-8' -ethyl carotene in the sample solution to be detected, V is the volume of the solution used for preparing the sample solution to be detected, and m is the mass of the feed or additive.
2. The method for detecting ethyl beta-apo-8' -carotene according to claim 1, wherein in the step (1), the sample solution to be detected of the feed sample is concentrated, specifically: taking 5ml of supernatant solution from a 100ml volumetric flask into a centrifuge tube, blow-drying by a nitrogen blowing instrument, and transferring 1ml of supernatant solution into a centrifuge tube with a volume ratio of 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution is re-dissolved and filtered to obtain a sample solution to be tested of a feed sample.
3. The method for detecting beta-apo-8' -carotene ethyl ester according to claim 2, wherein the mass of the weighed feed is 2.0g.
4. The method for detecting beta-apo-8' -carotene ethyl ester according to claim 1, wherein in the step (1), the sample solution to be detected of the additive sample is diluted, and the specific operations are as follows: taking 5ml of supernatant solution from a 100ml volumetric flask, and placing the supernatant solution into a 100mL volumetric flask with the volume ratio of 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution to a fixed volume, and filtering to obtain a sample solution to be tested of the additive sample.
5. The method for detecting ethyl beta-apo-8' -carotene according to claim 4, wherein the mass of the additive is 0.05g.
6. The method for detecting beta-apo-8' -carotene ethyl ester according to claim 1, wherein the conditions of the high performance liquid chromatograph are: adopting a C30 column, wherein the detection wavelength is 466nm, the column temperature is 30-35 ℃, and the mobile phase is 15-30:70-85 methyl tertiary butyl ether-methanol mixed solution; the flow rate of the elution is 0.8-1.5 ml/min.
7. The method for detecting beta-apo-8' -carotene ethyl ester according to claim 6, wherein the column temperature is 35 ℃, and the mobile phase is 15 in volume ratio: 85 methyl tert-butyl ether-methanol mixed solution; the flow rate of elution is 1.0ml/min, and the sample injection amount is 50 mu L; run time was 20min.
8. The method for detecting beta-apo-8' -carotene ethyl ester according to any one of claims 1-7, wherein the standard curve linear regression equation is: y= 479.074865x-1.4006713, y is the peak area of beta-apo-8 '-carotin acid ethyl ester, x is the mass concentration of beta-apo-8' -carotin acid ethyl ester in the sample solution to be detected, and the correlation coefficient R 2 =1.00000.
9. The method for detecting beta-apo-8 '-carotene ethyl ester according to any one of claims 1-7, wherein the lower limit of detection of the content of beta-apo-8' -carotene ethyl ester in the feed is 0.15mg/kg.
10. The method for detecting beta-apo-8 '-carotene ethyl ester according to claim 9, wherein the quantitative detection concentration of the beta-apo-8' -carotene ethyl ester content in the feed is 0.4mg/kg.
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