Method for detecting content of astaxanthin in animal serum

Abstract

The invention provides a method for detecting the content of astaxanthin in animal serum, and belongs to the technical field of chemical detection. The detection method provided by the invention comprises the following steps: mixing animal serum with an organic solvent, oscillating, centrifuging, and concentrating and filtering supernate obtained by centrifuging to obtain a to-be-detected solution; 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 1: 4. According to the method, the methane and the dichloromethane are used for dissolving the astaxanthin in the animal tissue sample, so that the extraction effect of the extracting solution on the astaxanthin in the sample is improved, and the detection accuracy of 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 astaxanthin content in the animal tissue is good, the time is saved, and the 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 animal serum. 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.

[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 animal serum. The method provided by the present invention can quickly, simply and accurately determine the astaxanthin content in animal serum.

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

[0008] The animal serum is mixed with an organic solvent, shaken, and centrifuged, and then the supernatant obtained by centrifugation is concentrated and 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 1:4.

[0011] In the present invention, when the volume ratio of methanol to dichloromethane is 1:4, the sample solubility and extraction purity will be higher. If the ratio changes, the concentration of the extracted sample will decrease and the dissolution will be incomplete.

[0012] In some embodiments of the present invention, the animal serum is broiler serum.

[0013] In some embodiments of the present invention, the volume ratio of the animal serum to the organic solvent is 0.1:(1-1.5).

[0014] In some embodiments of the present invention, the oscillation is oscillation in an oscillator or ultrasonic oscillation; when the oscillation is oscillation in an oscillator, the frequency of the oscillation is 30-60 Hz, preferably 30 Hz, and the time is 1-2 min, preferably 1 min; when the oscillation is performed in the oscillator, it also includes adding steel balls to the mixed solution of animal serum and organic solvent.

[0015] When the oscillation is ultrasonic oscillation, the frequency of the ultrasound is 50 Hz and the duration is 10 minutes.

[0016] The function of ultrasonic oscillation is to dissolve by vibration, thereby improving the reaction efficiency and effect.

[0017] In some embodiments of the present invention, the centrifugation condition is: centrifugation at 14000 r / min at 4°C for 5 min.

[0018] The invention ensures that the extracted sample will not be inactivated due to high temperature by low temperature centrifugation at 4°C.

[0019] In some embodiments of the present invention, the concentration comprises: blowing the supernatant liquid with nitrogen to dryness and then re-dissolving it with methanol.

[0020] In some embodiments of the present invention, the filtration is performed using a 0.22 μm filter membrane.

[0021] In some embodiments of the present invention, the parameters of the LC-MS / MS are: mobile phase A is a mixed solution of formic acid and water in a volume ratio of 1:99, mobile phase B is a mixed solution of formic acid and acetonitrile in a volume ratio of 1:99, the flow rate of mobile phase A and mobile phase B is 0.3 μL / min, the elution mode is isocratic elution, the chromatographic column is HSS T3, the column temperature of the chromatographic column is 40°C, and the injection volume is 2 μL.

[0022] In some embodiments of the present invention, the specifications of the chromatographic column are: 1.8 μm, 2.1×100 mm.

[0023] The present invention discloses the following technical effects:

[0024] The invention uses methane and dichloromethane to dissolve astaxanthin in an animal tissue sample, thereby improving the extraction effect of the extract on astaxanthin in the sample, thereby improving the accuracy of astaxanthin content detection.

[0025] 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 animal tissues, saves time and has simple operation steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is the chromatogram of the astaxanthin standard (10 ng / mL) in Example 1.

[0028] Figure 2 This is the chromatogram of broiler serum 1 in Example 2. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] 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.

[0035] All reagents used in the examples were of analytical grade.

[0036] The instruments and reagents used in the examples are shown in Tables 1 and 2.

[0037] Table 1 Experimental instruments and equipment

[0038]

[0039] Table 2 Experimental consumables and reagents

[0040] Reagent name Specifications / Article Number factory Standards CAS: 472-61-7 Anpu Methanol GB / T683-2006 Tongguang Fine Chemicals Company Dichloromethane G81014P GENERAL-REAGENT

[0041] Example 1

[0042] Plotting a standard curve

[0043] 1) Accurately weigh astaxanthin standard, dissolve it in acetone, and prepare a 100 μg / mL standard stock solution.

[0044] 2) Take the standard stock solution and use methanol to prepare a standard series of solutions with concentrations of 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0045] 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 = 1.583e 2 x-1.413e 3 , correlation coefficient (R 2) is 0.9983. The chromatographic conditions are as follows: the wavelength of liquid chromatography detection is 478nm; the mobile phase A of liquid chromatography detection is a mixed solution of formic acid and water in a volume ratio of 1:99, the mobile phase B is a mixed solution of formic acid and acetonitrile in a volume ratio of 1:99, the flow rate of mobile phase A and mobile phase B are both 0.3μL / min; the elution mode is isocratic elution; the chromatographic column is HSS T3 (1.8μm, 2.1×100mm) column; the column temperature of chromatographic detection is 40°C; the injection volume is 2μL.

[0046] Table 3 Standard astaxanthin determination liquid area

[0047]

[0048] Linear evaluation: Correlation coefficient R 2 The correlation coefficient R is 0.9983, 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.

[0049] Example 2

[0050] 1. Sample Extraction

[0051] 1) Take 100 μl of broiler serum and place it in a 2 mL centrifuge tube. Add 1 ml of organic solvent (V 甲醇 :V 二氯甲烷 =1:4), add steel balls, place in an oscillator and oscillate at 3 Hz for 1 min to obtain a slurry.

[0052] 2) The obtained slurry was centrifuged at 14000 r / min at 4°C for 5 min, and the supernatant was collected.

[0053] 3) After the supernatant was dried in a nitrogen blower, 1 ml of methanol was added to dissolve it, and then filtered using a 0.22 μm filter membrane. The filtrate was collected as the sample to be tested and stored in a brown injection bottle for LC-MS / MS analysis.

[0054] According to the test data of the astaxanthin standard obtained in Example 1, the astaxanthin content in the sample to be tested was measured (4 portions of broiler serum 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 .

[0055] Table 4 Astaxanthin content in broiler serum

[0056]

[0057] Note: The calculation formula is: X (μg / g) = c*V / 1000 / m

[0058] The meaning of each letter in the formula:

[0059] X: astaxanthin content in broiler serum (μg / g);

[0060] c: Concentration value (μg / mL) obtained by substituting the integrated peak area ratio in the sample into the standard curve;

[0061] V: volume of solution used for reconstitution (μL);

[0062] m: the weight of the sample (g).

[0063] 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 animal serum, characterized in that: The following steps are involved: The animal serum is mixed with an organic solvent, shaken, and centrifuged, and then the supernatant obtained by centrifugation is concentrated and 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 1:

4.

2. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The animal serum is broiler serum.

3. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The volume ratio of the animal serum to the organic solvent is 0.1:(1-1.5).

4. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The centrifugal conditions are: centrifugation at 14000 r / min at 4°C for 5 min.

5. The method for detecting astaxanthin content in animal serum according to claim 1, characterized in that: The concentration comprises: blowing the supernatant liquid with nitrogen to dryness, and then re-dissolving it with methanol.

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: mobile phase A is a mixed solution of formic acid and water in a volume ratio of 1:99, mobile phase B is a mixed solution of formic acid and acetonitrile in a volume ratio of 1:99, the flow rates of mobile phase A and mobile phase B are both 0.3 μL / min, the elution method is isocratic elution, the chromatographic column is HSS T3, the column temperature of the chromatographic column is 40°C, and the injection volume is 2 μL.

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