Method for detecting content of astaxanthin in broiler daily ration

Abstract

The invention provides a method for detecting the content of astaxanthin in broiler daily ration, and belongs to the technical field of chemical detection. The method provided by the invention comprises the following steps: mixing broiler daily ration with an organic solvent, oscillating, carrying out ultrasonic treatment, centrifuging, and filtering supernate obtained by centrifuging to obtain liquid to be detected; detecting the liquid to be detected by adopting LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry); the organic solvent is a mixed solution of methanol and dichloromethane in a volume ratio of 3: 1. According to the method, the methane and the dichloromethane are used for dissolving the astaxanthin in the broiler daily ration sample, so that the extraction effect of the extracting solution on the astaxanthin in the sample is improved, and the accuracy of detecting the content of the astaxanthin is improved. The method provided by the invention is simple, saves time, and also ensures the accuracy at the same time. Tests prove that the detection result of the content of the broiler chicken daily ration astaxanthin is good, meanwhile, time is saved, and operation steps are simple and convenient.

Description

Technical Field

[0001] The invention relates to the technical field of chemical detection, and in particular to a method for detecting astaxanthin content in broiler diets. Background Art

[0002] Astaxanthin's chemical name is: 3,3'-dihydroxy-4,4'diketo-β,β'carotene, a red solid powder, fat-soluble, insoluble in water, but soluble in organic solvents. Astaxanthin is widely present in the biological world, especially in aquatic animals such as shrimp, crab, fish and feathers of birds; it can also be obtained from algae and microorganisms.

[0003] Molecular structure of astaxanthin: The conjugated double bond system makes it appear bright red under visible light; the hydroxyl and keto groups give astaxanthin a certain water solubility and unique chemical properties; the molecular structure of astaxanthin consists of two chiral carbons at the 3 and 3' positions of the two terminal β-keto groups, which can produce three optical isomers: 3S, 3'S, 3R, 3'S, (3R, 3'R)-AX.

[0004] The molecular structure of astaxanthin enables it to stably capture free radicals, thereby preventing oxidative stress from damaging cells. In addition to anti-oxidation, astaxanthin also has a variety of biological activities such as anti-inflammatory, anti-cancer, and anti-fatigue, and is widely used in health products, cosmetics, feed, and other fields. Due to the excellent efficacy of astaxanthin, it has been widely used, including in the livestock industry.

[0005] Spectrophotometry can be used to accurately and quantitatively analyze the content of astaxanthin; high performance liquid chromatography has the advantages of fast separation speed and high online detection sensitivity, and is a determination method specified by the NFIA (National Feed Industry Association) of the United States. Due to the excellent efficacy of astaxanthin, it has been widely used, including in the animal husbandry industry. Therefore, the present invention aims to establish a simpler, faster and more effective method for detecting the content of astaxanthin in animal serum. Summary of the invention

[0006] Based on the above content, the purpose of the present invention is to provide a method for detecting the astaxanthin content in broiler diets. The detection method provided by the present invention can quickly, simply and accurately determine the astaxanthin content in broiler diets.

[0007] In order to achieve the above object, the present invention provides the following technical solution: a method for detecting astaxanthin content in broiler diets, comprising the following steps:

[0008] The broiler diet is mixed with an organic solvent, and then shaken, ultrasonicated, and centrifuged, and then the supernatant obtained by centrifugation is filtered to obtain a test solution;

[0009] Using LC-MS / MS to detect the test liquid;

[0010] The organic solvent is a mixed solution of methanol and dichloromethane in a volume ratio of 3:1.

[0011] Methanol makes the sample dissolve more dispersed and permeable, while dichloromethane makes the sample dissolve more fully. If the ratio of methanol to dichloromethane changes, the concentration of the extracted sample will change accordingly. When the organic solvent is a mixed solution of methanol and dichloromethane with a volume ratio of 3:1, the astaxanthin extraction effect is better.

[0012] In some embodiments of the present invention, the mass volume ratio of the broiler diet to the organic solvent is 0.2 g: 2-2.5 mL. When weighing the broiler diet, the mass of the broiler diet is accurate to 0.0001 g.

[0013] In some embodiments of the present invention, the frequency of the oscillation is 30 Hz and the duration is 1 min. During the oscillation process, steel balls are added to the mixture of the broiler diet and the organic solvent to make the grinding more complete.

[0014] In some embodiments of the present invention, the frequency of the ultrasound is 50 Hz, the duration is 60 min, and the temperature is room temperature. The function of ultrasonic oscillation is to dissolve by oscillation, thereby improving the reaction efficiency and effect.

[0015] In some embodiments of the present invention, the centrifugation condition is: centrifugation at 4000 r / min for 10 min at 4° C. The present invention uses low-temperature centrifugation at 4° C. to ensure that the extracted sample will not be inactivated due to high temperature.

[0016] In some embodiments of the present invention, the filtration is performed using a 0.22 μm filter membrane.

[0017] In some embodiments of the present invention, the parameters of the LC-MS / MS are: the mobile phase is a mixed solution of methanol and water in a volume ratio of 95:5, the flow rate of the mobile phase is 1 mL / min, the elution method is isocratic elution, the chromatographic column is Spursil C18, the column temperature of the chromatographic column is 25°C, and the injection volume is 20 μL.

[0018] Methanol, acetonitrile, tetrahydrofuran, water, dichloromethane, acetone, etc. are the most commonly used mobile phases in high performance liquid chromatography. In reverse phase chromatography, components with high polarity flow out of the column first due to their small K value, and components with low polarity flow out later. As the proportion of organic solvent in the mobile phase increases, the polarity of the mobile phase decreases, and the elution power increases. Therefore, using a mixed solution of methanol and water with a volume ratio of 95:5 as the mobile phase can improve the elution power and is more economical.

[0019] In some embodiments of the present invention, the chromatographic column has a specification of 250*4.6 mm and 5 μm.

[0020] The present invention discloses the following technical effects:

[0021] The present invention uses methane and dichloromethane to dissolve astaxanthin in animal diet samples, thereby improving the extraction effect of the extract on astaxanthin in the sample, thereby improving the accuracy of astaxanthin content detection.

[0022] The method provided by the invention is simple, time-saving and accurate. Experiments have shown that the method has good results in detecting the astaxanthin content in broiler diets, saves time and has simple operation steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Chromatogram of the astaxanthin standard (5 mg / mL) in Example 1.

[0025] Figure 2 Chromatogram of broiler diet 1 in Example 2. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Room temperature in the present invention means a temperature of 20-30°C.

[0032] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0033] All reagents used in the examples were of analytical grade.

[0034] The instruments and reagents used in the examples are shown in Tables 1 and 2.

[0035] Table 1 Experimental instruments and equipment

[0036]

[0037] Table 2 Experimental consumables and reagents

[0038] Reagent name Specifications / Article Number factory Standards CAS: 472-61-7 Anpu Methanol GB / T683-2006 Tongguang Fine Chemicals Company Dichloromethane G81014P GENERAL-REAGENT

[0039] Example 1

[0040] Plotting a standard curve

[0041] 1) Accurately weigh astaxanthin standard, dissolve it in acetone, and prepare a 100 μg / mL standard stock solution.

[0042] 2) Take the standard stock solution and use methanol to prepare a standard series of solutions with concentrations of 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, and 50 mg / L.

[0043] The astaxanthin standard working solution was injected and analyzed by HPLC according to the chromatographic conditions provided below. The concentration and peak area of ​​the standard solution are shown in Table 3 and the spectrum is shown in Figure 1 , its linear regression equation is y = 97.242x + 199.3, and the correlation coefficient (R 2) is 0.9968. The chromatographic conditions are as follows: the wavelength of the liquid chromatography detection is 478 nm; the mobile phase of the liquid chromatography detection is a mixed solution of methanol and water in a volume ratio of 95:5, and the flow rate of the mobile phase is 1 mL / min; the elution method is isocratic elution; the chromatographic column is Spursil C18 (250*4.6 mm; 5 μm); the column temperature of the chromatographic detection is 25°C; and the injection volume is 20 μL.

[0044] Table 3 Standard astaxanthin determination liquid area

[0045]

[0046] Linear evaluation: Correlation coefficient R 2 The correlation coefficient R is 0.9968, which meets the requirements of GB / T27404 "Laboratory Quality Control Specification for Food Physical and Chemical Testing" (GB / T 27404 requires that the correlation coefficient R 2 >0.99), so it is feasible to determine the astaxanthin content using this method.

[0047] Example 2

[0048] 1. Sample Extraction

[0049] 1) Take 0.2000g of broiler diet and place it in a 15mL centrifuge tube. Add 2ml of organic solvent (V 甲醇 :V 二氯甲烷 =3:1), add steel balls and place in an oscillator to oscillate at 3 Hz for 1 min, then ultrasonicate at 50 Hz for 60 min at room temperature to obtain a slurry.

[0050] 2) The obtained slurry was centrifuged at 4000 r / min at 4°C for 10 min, and the supernatant was collected.

[0051] 3) Filter the obtained supernatant using a 0.22 μm filter membrane, collect the filtrate as the sample to be tested, and store it in a brown injection bottle for LC-MS / MS analysis.

[0052] According to the test data of the astaxanthin standard obtained in Example 1, the astaxanthin content in the sample to be tested was measured (two portions of broiler diet were taken to prepare the sample to be tested). The astaxanthin determination method provided by the present invention meets the methodological verification requirements and can accurately determine the astaxanthin content. The astaxanthin content results are shown in Table 4 and the spectra are shown in Figure 2 .

[0053] Table 4 Astaxanthin content in broiler diets

[0054]

[0055] Note: The calculation formula is: X (μg / g) = c*V / 1000 / m

[0056] The meaning of each letter in the formula:

[0057] X: astaxanthin content in broiler diets (μg / g);

[0058] c: Concentration value (μg / mL) obtained by substituting the integrated peak area ratio in the sample into the standard curve;

[0059] V: volume of organic solvent extract used (μL);

[0060] m: the weight of the sample (g).

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting astaxanthin content in broiler diets, characterized in that: The following steps are involved: The broiler diet is mixed with an organic solvent, and then shaken, ultrasonicated, and centrifuged, and then the supernatant obtained by centrifugation is filtered to obtain a test solution; Using LC-MS / MS to detect the test liquid; The organic solvent is a mixed solution of methanol and dichloromethane in a volume ratio of 3:

1.

2. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The mass volume ratio of the broiler diet to the organic solvent is 0.2g:2-2.5mL.

3. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The frequency of the oscillation is 30 Hz and the duration is 1 min.

4. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The frequency of the ultrasound is 50 Hz, the time is 60 min, and the temperature is room temperature.

5. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The centrifugal conditions are: centrifugation at 4°C and a rotation speed of 4000 r / min for 10 min.

6. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The filtration is carried out using a 0.22 μm filter membrane.

7. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The parameters of the LC-MS / MS are as follows: the mobile phase is a mixed solution of methanol and water in a volume ratio of 95:5, the flow rate of the mobile phase is 1 mL / min, the elution mode is isocratic elution, the chromatographic column is Spursil C18, the column temperature of the chromatographic column is 25°C, and the injection volume is 20 μL.

Images