Rapid detection method for astaxanthin in broiler daily ration

Abstract

The invention relates to the technical field of quantitative detection, and discloses a rapid detection method for astaxanthin in broiler daily ration, which comprises the following steps: sequentially adopting triethylamine, 4-aminophenyl urea and ascorbyl palmitate to carry out surface modification treatment on acid-activated silica gel microspheres to obtain an astaxanthin specific bonding material; filling the bonding material into an extraction column to prepare a solid-phase extraction column; the method comprises the following steps: grinding a broiler daily ration sample, mixing with acetonitrile, oscillating, extracting, and centrifuging to take supernate; activating the solid-phase extraction column, loading a sample, leaching and eluting to obtain an eluent; and carrying out nitrogen gas blow-drying on the eluent, redissolving with methanol, detecting by adopting high performance liquid chromatography, and calculating to obtain the content of astaxanthin in the broiler daily ration. Through ureido specific recognition, antioxidant protection and silicon hydroxyl sealing bifunctional bonding material design, the problems of contradiction between accuracy and rapidness, poor specificity and weak applicability of a traditional method are effectively solved, the material can be reused, and multi-scene detection requirements are met.

Description

Technical Field

[0001] This invention relates to the field of quantitative detection technology, and more specifically, to a rapid detection method for astaxanthin in broiler diets. Background Technology

[0002] Astaxanthin is a natural carotenoid with strong antioxidant, immunomodulatory, and coloring properties. It is widely used as a functional additive in broiler diets. The amount added directly determines the color quality of broiler skin, muscle, and eggs, and also affects breeding cost control and product market competitiveness. Therefore, establishing a precise and rapid detection method for astaxanthin in broiler diets is of great significance for feed quality control and breeding production guidance.

[0003] Currently, the main methods for detecting astaxanthin include high-performance liquid chromatography (HPLC), colorimetry, immunochromatography, and near-infrared spectroscopy. Among them, HPLC is the mainstream standard method for astaxanthin detection due to its high separation efficiency and accurate quantification (such as GB / T 23745-2009 and AOAC related standards). However, it has significant limitations in the detection process: broiler diets have complex matrices containing various interfering components such as corn, soybean meal, premixes, lutein, and β-carotene. Traditional HPLC detection requires sample purification through pretreatment steps such as liquid-liquid extraction and conventional solid-phase extraction (SPE). However, existing SPE materials (such as unmodified silica gel and C18 adsorbent) have weak specific adsorption capacity for astaxanthin, making it difficult to effectively separate interfering pigments and impurities in the matrix. This results in large fluctuations in the recovery rate (usually 70%-95%) and poor reproducibility. Furthermore, traditional pretreatment processes are cumbersome (requiring multiple extractions, centrifugation, and concentration), time-consuming (single sample pretreatment ≥60 min), and dependent on laboratory equipment, which cannot meet the rapid sampling needs of farms and feed distributors.

[0004] To adapt to various detection scenarios, researchers have developed rapid techniques such as colorimetry and immunochromatography. Colorimetry, based on the colorimetric reaction of astaxanthin, is simple to operate and low in cost, but it has poor specificity and is easily interfered with by other carotenoids, proteins, and other components in the diet. Its detection limit is only about 0.02 mg / g, which is insufficient to meet the precise detection requirements of diets with low astaxanthin addition levels. Immunochromatography achieves rapid screening through specific antigen-antibody binding, with a detection time ≤15 min. However, the test strips have poor stability (requiring refrigeration), high cost for batch testing, and insufficient quantitative accuracy (relative standard deviation RSD ≥8%), making it unable to replace the accurate quantitative function of HPLC. Near-infrared spectroscopy can achieve non-destructive and rapid detection, but the instrument cost is high (portable equipment ≥50,000 RMB), the model is easily affected by differences in diet formulation, limiting its applicability, and the detection limit (0.03 mg / g) is higher than the actual detection requirements.

[0005] In the field of solid-phase extraction materials, while existing technologies have attempted to use silica microspheres as carriers for surface modification, they suffer from several drawbacks: First, the modifying groups are often limited to a single type, typically introducing only hydrophobic or simple coordinating groups, lacking specific recognition sites for astaxanthin and thus failing to achieve targeted enrichment. Second, astaxanthin is easily oxidized and degraded during detection, and existing materials do not incorporate antioxidant protection, leading to target analyte loss during the detection process. Third, residual silanol groups on the surface of silica microspheres can easily trigger non-specific adsorption, further reducing purification efficiency and detection accuracy. Therefore, developing astaxanthin-binding materials that combine high specificity, antioxidant properties, and stability is crucial to addressing the complex pretreatment and poor applicability of traditional HPLC detection methods.

[0006] Furthermore, with the large-scale and intensive development of the livestock industry, the market demand for quality control of broiler feed is becoming increasingly urgent—feed producers need to quickly verify the accuracy of raw material additions, and farms need to monitor feed quality in real time. Existing technologies cannot simultaneously meet the dual requirements of "rapid operation" and "precise quantification," necessitating a rapid detection method with simplified pretreatment, high specificity, and accurate detection to fill the technological gap and provide technical support for the quality control of astaxanthin in broiler feed. Summary of the Invention

[0007] In view of this, the present invention proposes a rapid detection method for astaxanthin in broiler diets, aiming to solve the problem in the current technology that the astaxanthin content in broiler diets cannot be quickly and accurately quantified due to the complexity of the broiler diet matrix.

[0008] This invention proposes a rapid detection method for astaxanthin in broiler diets, comprising the following steps: 1) Activated silica microspheres are obtained by mixing silica microspheres with hydrochloric acid solution and carrying out a hydrolysis reaction; 2) Under anaerobic conditions, activated silica microspheres are mixed with dichloromethane and triethylamine to carry out a preliminary reaction to obtain surface-modified silica microspheres. Then, the surface-modified silica microspheres are mixed with a mixed solvent and 4-aminophenylurea to carry out a grafting reaction to obtain silica microspheres with urea groups on the surface. 3) The silica microspheres with urea groups on the surface are mixed and reacted with ascorbate palmitate and organic solvent to introduce antioxidant groups on the surface of the silica microspheres. Trimethylchlorosilane is added to the reaction system to continue the reaction and block the residual silanol groups to obtain astaxanthin-specific bonding materials. 4) Take a sample of broiler feed, grind it, mix it with acetonitrile, shake and extract, centrifuge and collect the supernatant; The bonding material was loaded into the extraction column, and the extraction column was successively washed with ethanol and purged with nitrogen to obtain a solid-phase extraction column. 5) The solid-phase extraction column was activated sequentially with methanol and n-hexane, and then the sample was loaded, washed, and eluted to obtain the eluent; 6) After drying the eluent with nitrogen, it was reconstituted with methanol and detected by high performance liquid chromatography to calculate the astaxanthin content in the broiler diet.

[0009] Preferably, the silica microspheres in step 1) have a particle size of 50-150 μm and a specific surface area of ​​200-400 m². 2 / g; The concentration of the hydrochloric acid solution is 0.5-2 mol / L.

[0010] Preferably, the solid-liquid ratio of the silica microspheres to the hydrochloric acid solution in step 1) is 1g:10-20mL; The hydrolysis reaction temperature is 60-80℃, and the reaction time is 2-4h.

[0011] Preferably, the ratio of activated silica microspheres, dichloromethane, and triethylamine in step 2) is 1g: 15-30mL: 2-5mL; In step 2), the reaction temperature for modifying the surface of the silica microspheres is 25-35℃, and the reaction time is 1-3h.

[0012] Preferably, the mixed solvent in step 2) is composed of dichloromethane and anhydrous ethanol in a volume ratio of 3-5:1; The mass ratio of the surface-modified silica microspheres to the mixed solvent and 4-aminophenylurea is 1:5-10:0.3-0.8; The grafting reaction is carried out at a temperature of 40-60℃ for 3-6 hours.

[0013] Preferably, in step 3), the mixing ratio of the urea-based silica microspheres on the surface to ascorbate palmitate and organic solvent is 1g:0.1-0.3:10-15mL, the mixing reaction temperature is 50-70℃, and the time is 2-4h. The amount of trimethylchlorosilane added is 5%-10% of the total mass of the reaction system. After adding trimethylchlorosilane, the reaction temperature is controlled at 25-35℃ and the reaction time is 1-2h.

[0014] Preferably, the particle size of the broiler diet sample after grinding in step 4) is 40-80 mesh, the mixing ratio of the broiler diet sample and acetonitrile is 1g: 5-10mL, the shaking extraction speed is 150-250r / min, and the extraction time is 10-20min. The centrifugation speed is 8000-12000 r / min, and the centrifugation time is 5-10 min; The extraction column has a column volume of 3-6 mL, a bonding material loading of 0.1-0.3 g, an ethanol washing volume of 2-5 mL, a washing flow rate of 1-2 mL / min, a nitrogen purging pressure of 0.1-0.3 MPa, and a purging time of 3-5 min.

[0015] Preferably, the activation volume of methanol in step 5) is 2-5 mL, and the activation flow rate is 0.5-1 mL / min; The activation volume of the n-hexane is 2-5 mL, and the activation flow rate is 0.5-1 mL / min.

[0016] Preferably, the sample loading flow rate in step 5) is 0.3-0.8 mL / min, and the sample loading volume is 1-5 mL; The rinsing agent used in the rinsing process is a mixture of n-hexane and ethyl acetate, with a volume ratio of n-hexane to ethyl acetate of 8-10:1; The rinsing volume is 3-6 mL, and the rinsing flow rate is 0.5-1 mL / min; The eluent used in the elution process is a mixture of methanol and dichloromethane, with a volume ratio of 1-3:1. The amount of eluent used in the elution process is 2-5 mL, and the flow rate of the eluent is 0.3-0.6 mL / min.

[0017] Preferably, the parameters for high-performance liquid chromatography in step 6) are as follows: The chromatographic column was a C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The mobile phase is a mixed solution of methanol, acetonitrile, and dichloromethane, with a volume ratio of 40-60:30-50:5-15. The flow rate of the mobile phase is 0.8-1.2 mL / min; The column temperature of the chromatographic column is 25-35℃; The detection wavelength is 470±2nm; The injection volume is 10-20 μL; The astaxanthin content was calculated using the external standard method.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention introduces urea groups through 4-aminophenylurea grafting. These urea groups form specific hydrogen bonds with the hydroxyl and ketone groups in the astaxanthin molecule, achieving targeted enrichment of astaxanthin with a cross-adsorption rate of ≤3% for lutein and β-carotene. Trimethylchlorosilane is used to seal residual silanol groups on the surface of the silica microspheres, preventing non-specific adsorption and further reducing matrix interference. During the preparation of the bonding material, ascorbate palmitate grafting introduces antioxidant groups, which provide protection for astaxanthin throughout sample processing, inhibiting oxidative degradation.

[0019] The solid-phase extraction material prepared by this invention can be stored for a long time and used multiple times. After simple grinding, the sample is extracted by shaking with acetonitrile, the supernatant is obtained by centrifugation, and the sample is directly loaded onto the pre-made extraction column. The purification is completed through four steps: "activation-loading-rinsing-elution". The entire pretreatment process takes ≤30 minutes. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0021] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention proposes a rapid detection method for astaxanthin in broiler diets, comprising the following steps: 1) Activated silica microspheres are obtained by mixing silica microspheres with hydrochloric acid solution and carrying out a hydrolysis reaction; 2) Under anaerobic conditions, activated silica microspheres are mixed with dichloromethane and triethylamine to carry out a preliminary reaction to obtain surface-modified silica microspheres. Then, the surface-modified silica microspheres are mixed with a mixed solvent and 4-aminophenylurea to carry out a grafting reaction to obtain silica microspheres with urea groups on the surface. 3) The silica microspheres with urea groups on the surface are mixed and reacted with ascorbate palmitate and organic solvent to introduce antioxidant groups on the surface of the silica microspheres. Trimethylchlorosilane is added to the reaction system to continue the reaction and block the residual silanol groups to obtain astaxanthin-specific bonding materials. 4) Take a sample of broiler feed, grind it, mix it with acetonitrile, shake and extract, centrifuge and collect the supernatant; The bonding material was loaded into the extraction column, and the extraction column was successively washed with ethanol and purged with nitrogen to obtain a solid-phase extraction column. 5) The solid-phase extraction column was activated sequentially with methanol and n-hexane, and then the sample was loaded, washed, and eluted to obtain the eluent; 6) After drying the eluent with nitrogen, it was reconstituted with methanol and detected by high performance liquid chromatography to calculate the astaxanthin content in the broiler diet.

[0026] In step 1), a small number of silanol groups are initially present on the surface of the silica microspheres. Hydrochloric acid, as a strong acid, catalyzes the hydrolysis and breakage of siloxane bonds, generating a large number of free silanol groups. At the same time, moderate hydrolysis can form a porous structure on the surface of the microspheres, which not only preserves the integrity of the particles but also increases the specific surface area.

[0027] In the first preliminary reaction of step 2), triethylamine acts as an acid-binding agent, promoting the nucleophilic substitution reaction between the silanol groups on the surface of the activated silica microspheres and dichloromethane, introducing a chloromethyl active functional group. In the second grafting reaction, the chloromethyl group undergoes a nucleophilic substitution reaction with the amino group of 4-aminophenylurea, covalently bonding the urea group to the silica surface. The hydrogen atoms in the urea group can form specific hydrogen bonds with the hydroxyl and ketone groups in the astaxanthin molecule, achieving targeted adsorption of astaxanthin, while exhibiting extremely weak adsorption capacity for interfering pigments such as lutein and β-carotene, which lack complementary hydrogen bond structures.

[0028] The phenolic hydroxyl groups in the ascorbate palmitate molecule used in step 3) have strong reducing properties. They can bind to the active sites next to the residual silanol groups or urea groups on the surface of silica gel through ester bonds, forming an antioxidant protective layer on the material surface and inhibiting the oxidation reaction of astaxanthin during the extraction and purification process. Trimethylchlorosilane, as a silanizing agent, can undergo a substitution reaction with unreacted residual silanol groups to generate inert trimethylsiloxy groups, eliminating the polar adsorption of silanol groups and avoiding the non-specific adsorption of polar impurities such as proteins and carbohydrates.

[0029] Acetonitrile is used as the extraction solvent because astaxanthin is a fat-soluble compound, and acetonitrile, as a highly polar organic solvent, can efficiently dissolve astaxanthin. Simultaneously, it can disrupt the binding between astaxanthin and protein in broiler feed, promoting the release of astaxanthin from the matrix. Grinding the sample increases the contact area between the sample and the extractant. Shaking and centrifugation work together to remove solid impurities (such as cellulose and minerals), resulting in a clear extract. After the bonded material is packed into the extraction column, ethanol washing removes residual preparation process impurities (such as unreacted reagents) from the material surface, and nitrogen purging removes residual moisture and ethanol from the column, preventing interference with subsequent adsorption.

[0030] During the activation of the extraction column, methanol, as a polar solvent, swells the porous structure of the bonded material, fully exposing the urea groups and increasing the adsorption capacity. Hexane adjusts the surface polarity of the material, matching its hydrophobicity with that of astaxanthin and enhancing the adsorption force. During sample loading, astaxanthin in the extract specifically binds to the urea groups via hydrogen bonds and is firmly adsorbed onto the material surface, while most interfering impurities (such as lutein and carbohydrates) remain in a free state because they cannot form effective hydrogen bonds. The eluent, with its low polarity, can wash away unadsorbed interfering impurities without disrupting the hydrogen bonds between astaxanthin and urea groups; the more polar eluent competitively breaks these hydrogen bonds, causing rapid desorption of astaxanthin and achieving the enrichment and separation of the target analyte.

[0031] Nitrogen purging rapidly removes volatile solvents (methanol, dichloromethane) from the eluent, concentrating the target analyte. After methanol redissolution, the sample solution exhibits good compatibility with the HPLC mobile phase, avoiding peak distortion caused by solvent effects. The C18 column is a reversed-phase column. Astaxanthin, being a lipid-soluble compound, exhibits stronger hydrophobic interactions with the stationary phase (C18 chain) than with interfering substances. With an optimized mobile phase ratio (methanol-acetonitrile-dichloromethane), baseline separation from trace residual interfering substances can be achieved. The characteristic absorption peak of astaxanthin is 470±2 nm; detection at this wavelength yields the highest sensitivity and specificity, avoiding false positives. The external standard method achieves quantitative calculation by comparing the sample peak area with a standard curve; the linearity of the standard curve directly determines the accuracy of the quantification.

[0032] In this invention, the silica microspheres mentioned in step 1) have a particle size of 50-150 μm to ensure permeability when packing the extraction column and avoid clogging; and a specific surface area of ​​200-400 m². 2 / g provides ample reaction sites.

[0033] The concentration of the hydrochloric acid solution is 0.5-2 mol / L, preferably 0.8-1.8 mol / L, more preferably 1-1.5 mol / L, and even more preferably 1.2 mol / L.

[0034] In this invention, the solid-liquid ratio of the silica microspheres to the hydrochloric acid solution in step 1) is 1g:10-20mL, preferably 1g:12-18mL, more preferably 1g:14-16mL, and even more preferably 1g:15mL.

[0035] In this invention, the hydrolysis reaction temperature is 60-80℃, preferably 63-78℃, more preferably 65-75℃, and even more preferably 68-72℃; the reaction time is 2-4h, preferably 2.5-3.5h, and even more preferably 3h.

[0036] In this invention, the ratio of activated silica microspheres, dichloromethane, and triethylamine in step 2) is 1g:15-30mL:2-5mL, preferably 1g:18-27mL:2.5-4.5mL, more preferably 1g:20-25mL:3-4mL, and even more preferably 1g:22mL:3.5mL.

[0037] In this invention, the reaction temperature for modifying the surface of the silica microspheres in step 2) is 25-35℃, preferably 27-32℃, and more preferably 30℃; the reaction time is 1-3h, preferably 1.5-2.5h, and more preferably 2h.

[0038] In this invention, the mixed solvent in step 2) is composed of dichloromethane and anhydrous ethanol in a volume ratio of 3-5:1, preferably 3.5-4.5:1, more preferably 3.8-4.2:1, and even more preferably 4:1.

[0039] In this invention, the mass ratio of the surface-modified silica microspheres to the mixed solvent and 4-aminophenylurea is 1:5-10:0.3-0.8, preferably 1:6-9:0.4-0.7, and more preferably 1:7-8:0.5-0.6.

[0040] In this invention, the temperature of the grafting reaction is 40-60°C, preferably 43-58°C, more preferably 45-55°C, and even more preferably 48-52°C; the time is 3-6 hours, preferably 3.5-5.5 hours, more preferably 4-5 hours, and even more preferably 4.5 hours.

[0041] In this invention, the mixing ratio of the urea-based silica microspheres on the surface, ascorbate palmitate, and organic solvent in step 3) is 1g:0.1-0.3:10-15mL, preferably 1g:0.13-0.28:11-14mL, more preferably 1g:0.15-0.25:12-13mL, and even more preferably 1g:0.2:12mL; the mixing reaction temperature is 50-70℃, preferably 55-65℃, more preferably 58-63℃, and even more preferably 60℃; the time is 2-4h, preferably 2.5-3.5h, and even more preferably 3h.

[0042] In this invention, the amount of trimethylchlorosilane added is 5%-10% of the total mass of the reaction system, preferably 6%-9%, and more preferably 7%-8%; after adding trimethylchlorosilane, the reaction temperature is controlled at 25-35℃, preferably 27-33℃, more preferably 29-31℃, and more preferably 30℃; the reaction time is 1-2h, preferably 1.25-1.75h, and more preferably 1.5h.

[0043] In this invention, the particle size of the broiler diet sample after grinding in step 4) is 40-80 mesh; the mixing ratio of the broiler diet sample and acetonitrile is 1g:5-10mL, preferably 1g:6-9mL, more preferably 1g:7-8mL; the shaking extraction speed is 150-250r / min, preferably 170-230r / min, more preferably 190-210r / min, more preferably 200r / min; the extraction time is 10-20min, preferably 12-18min, more preferably 14-16min, more preferably 15min.

[0044] In this invention, the centrifugation speed is 8000-12000 r / min, preferably 8000-12000 r / min, more preferably 9000-11000 r / min, and more preferably 10000 r / min; the centrifugation time is 5-10 min, preferably 6-9 min, and more preferably 7-8 min.

[0045] In this invention, the extraction column has a column volume of 3-6 mL, preferably 3.5-5.5 mL, more preferably 4-5 mL, and even more preferably 5.5 mL; the amount of bonding material is 0.1-0.3 g, preferably 0.15-0.25 g, more preferably 0.18-0.22 g, and even more preferably 0.2 g; the amount of ethanol used for rinsing is 2-5 mL, preferably 2.5-4.5 mL, more preferably 3-4 mL, and even more preferably 3.5 mL; the rinsing flow rate is 1-2 mL / min, preferably 1.2-1.8 mL / min, and even more preferably 1.5 mL / min; the nitrogen purging pressure is 0.1-0.3 MPa, preferably 0.15-0.25 MPa, and even more preferably 0.2 MPa; and the purging time is 3-5 min, which can be selected as 3 min, 4 min, or 5 min.

[0046] In this invention, the activation volume of methanol in step 5) is 2-5 mL, preferably 2.5-4.5 mL, and more preferably 3-4 mL; the activation flow rate is 0.5-1 mL / min, preferably 0.6-0.9 mL / min, and more preferably 0.7-0.8 mL / min.

[0047] In this invention, the activation volume of the n-hexane is 2-5 mL, preferably 2.5-4.5 mL, and more preferably 3-4 mL; the activation flow rate is 0.5-1 mL / min, preferably 0.6-0.9 mL / min, and more preferably 0.7-0.8 mL / min.

[0048] In this invention, the sample loading flow rate in step 5) is 0.3-0.8 mL / min, preferably 0.4-0.7 mL / min, more preferably 0.5-0.6 mL / min; the sample loading volume is 1-5 mL, preferably 2-4 mL, more preferably 2.5-3.5 mL, and more preferably 3 mL.

[0049] In this invention, the rinsing agent used in the rinsing process is a mixture of n-hexane and ethyl acetate, with a volume ratio of n-hexane to ethyl acetate of 8-10:1, preferably 8.3-9.8:1, more preferably 8.5-9.5:1, and even more preferably 9:1.

[0050] In this invention, the rinsing volume is 3-6 mL, preferably 3.5-5.5 mL, more preferably 4-5 mL, and even more preferably 5.5 mL; the rinsing flow rate is 0.5-1 mL / min, preferably 0.6-0.9 mL / min, and even more preferably 0.7-0.8 mL / min.

[0051] In this invention, the eluent used in the elution process is a mixture of methanol and dichloromethane, with a volume ratio of 1-3:1, preferably 1.5-2.5:1, more preferably 1.8-2.2:1, and even more preferably 2:1.

[0052] In this invention, the amount of eluent used in the elution process is 2-5 mL, preferably 2.5-4.5 mL, more preferably 3-4 mL, and even more preferably 3.5 mL; the flow rate of the eluent is 0.3-0.6 mL / min, preferably 0.4-0.5 mL / min, and even more preferably 0.45 mL / min.

[0053] In this invention, the parameters of the high-performance liquid chromatography (HPLC) in step 6) are as follows: The chromatographic column was a C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The mobile phase is a mixed solution of methanol, acetonitrile, and dichloromethane, with a volume ratio of 40-60:30-50:5-15. The flow rate of the mobile phase is 0.8-1.2 mL / min; The column temperature of the chromatographic column is 25-35℃; The detection wavelength is 470±2nm; The injection volume is 10-20 μL.

[0054] In this invention, the astaxanthin content is calculated using the external standard method.

[0055] The materials and samples involved in the following embodiments and comparative examples are as follows: Broiler diet samples: blank broiler diet (tested to be free of astaxanthin) and simulated diets with added astaxanthin (low concentration 0.02mg / g, medium concentration 0.05mg / g, high concentration 0.1mg / g), all of which are commercially available conventional broiler compound feeds (main components: corn 60%, soybean meal 25%, premix 10%, oil 5%). Reagents: Hydrochloric acid (analytical grade), dichloromethane, triethylamine, 4-aminophenylurea (purity ≥98%), ascorbate palmitate (purity ≥95%), trimethylchlorosilane, acetonitrile, methanol, n-hexane, ethyl acetate (all chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); astaxanthin standard (purity ≥98%, Sigma-Aldrich); Equipment: High-performance liquid chromatograph (Agilent 1260, equipped with C18 column 4.6mm×250mm, 5μm), portable shaker (HZQ-3, Harbin Donglian Electronic Technology Development Co., Ltd.), high-speed refrigerated centrifuge (Thermo ST16R), solid phase extraction device (Supelco Visiprep™), nitrogen purging device (DC-12, Shanghai Anpu Experimental Technology Co., Ltd.), electric thermostatic oven (DHG-9070A, Shanghai Jinghong Experimental Equipment Co., Ltd.), electronic balance (accuracy 0.001g, Mettler Toledo), standard test sieve (40 mesh).

[0056] Example 1 (1) Hydrolysis and activation of silica microspheres: The silica microspheres have a particle size of 100 μm and a specific surface area of ​​300 m². 2 / g; hydrochloric acid concentration 1.2mol / L; solid-liquid ratio 1g:15mL; hydrolysis temperature 70℃ (within the range of 68-72℃); reaction time 3h; (2) Surface modification and urea grafting: Activated silica microspheres: dichloromethane: triethylamine = 1g: 22mL: 3.5mL; initial modification temperature 30℃, time 2h; mixed solvent (dichloromethane: anhydrous ethanol = 4: 1); surface modified silica microspheres: mixed solvent: 4-aminophenylurea = 1: 7.5: 0.55 (mass ratio); grafting temperature 50℃ (within the range of 48-52℃), time 4.5h; (3) Introduction of antioxidant groups and silanol blocking: Urea-based silica microspheres: Ascorbate palmitate: Organic solvent (methanol) = 1g: 0.2g: 12mL; Mixing reaction temperature 60℃, time 3h; Trimethylchlorosilane addition amount 7.5% (total mass of reaction system); Blocking temperature 30℃, time 1.5h; (4) Sample extraction and solid-phase extraction column preparation: The sample (simulated diet with added astaxanthin) was ground to 60 mesh; sample: acetonitrile = 1g: 7.5mL; shaking speed 200r / min, extraction time 15min; centrifugation speed 10000r / min, time 7.5min; extraction column volume 5.5mL; bonding material loading 0.2g; ethanol washing volume 3.5mL, flow rate 1.5mL / min; nitrogen purging pressure 0.2MPa, time 4min; (5) Construction of the standard curve: Prepare astaxanthin standard solutions (solvent: methanol) with concentration gradients of 0.01-1.0 μg / mL (0.01 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.10 μg / mL, 0.50 μg / mL, 1.00 μg / mL). Use methanol as a blank control to subtract solvent background interference. Plot the concentration of the standard working solution (x, unit: μg / mL) as the abscissa and the corresponding average peak area (y, peak area × 10) as the y-axis.4 Using AU as the ordinate, a linear regression analysis was performed. The linear regression equation was y = 32.85x - 0.012, and the correlation coefficient R0 was [value missing]. 2 =0.9998, the HPLC parameters used in the standard curve plotting process are the same as those used in the detection process.

[0057] (6) Solid phase extraction purification: Methanol activation volume 3.5 mL, flow rate 0.75 mL / min; n-hexane activation volume 3.5 mL, flow rate 0.75 mL / min; sample loading flow rate 0.55 mL / min, sample loading volume 3 mL; eluent (n-hexane: ethyl acetate = 9:1), volume 5.5 mL, flow rate 0.75 mL / min; eluent (methanol: dichloromethane = 2:1), volume 3.5 mL, flow rate 0.45 mL / min; (7) HPLC detection: mobile phase (methanol:acetonitrile:dichloromethane = 50:40:10); flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 470 nm; injection volume 15 μL.

[0058] The test results of the three concentrations of simulated diets are shown in Table 1.

[0059] Table 1. Detection results of three concentrations of simulated diets

[0060] Example 2 (1) Activation by hydrolysis of silica microspheres Silica microspheres have a particle size of 80 μm and a specific surface area of ​​250 m². 2 / g; hydrochloric acid concentration 1.0mol / L (within the range of 1-1.5mL); solid-liquid ratio 1g:15mL; hydrolysis temperature 70℃ (within the range of 68-72℃); reaction time 3h; (2) Surface modification and urea grafting Activated silica microspheres: dichloromethane: triethylamine = 1g: 22mL: 3.5mL; initial modification temperature 30℃, time 2h; mixed solvent (dichloromethane: anhydrous ethanol = 4: 1); surface-modified silica microspheres: mixed solvent: 4-aminophenylurea = 1: 7.5: 0.55 (mass ratio); grafting temperature 50℃ (within the range of 48-52℃), time 4.5h; (3) Introduction of antioxidant groups and silanol blockade Urea-based silica microspheres: ascorbate palmitate: organic solvent (methanol) = 1g: 0.2g: 12mL; mixing reaction temperature 60℃, time 3h; trimethylchlorosilane addition amount 7.5% (total mass of reaction system); sealing temperature 30℃, time 1.5h; (4) Sample extraction and preparation of solid phase extraction column The sample (a simulated diet supplemented with astaxanthin) was ground to 60 mesh; the sample-acetonitrile ratio was 1 g: 7.5 mL; the shaking speed was 200 r / min, and the extraction time was 15 min; the centrifugation speed was 10000 r / min, and the time was 7.5 min; the extraction column volume was 5.5 mL; the bonding material packing amount was 0.2 g; the ethanol washing volume was 3.5 mL, and the flow rate was 1.5 mL / min; the nitrogen purging pressure was 0.2 MPa, and the time was 4 min. (5) Drawing the standard curve Prepare astaxanthin standard solutions (methanol as solvent) with a concentration gradient of 0.01-1.0 μg / mL. Use methanol as a blank control to subtract solvent background interference. Plot the concentration of the standard working solution (x, unit: μg / mL) on the x-axis and the corresponding average peak area (y, peak area × 10) on the y-axis. 4 Using AU as the ordinate, a linear regression analysis was performed. The linear regression equation was y = 32.78x - 0.015, and the correlation coefficient R0 was [value missing]. 2 =0.9997, the HPLC parameters used in the standard curve plotting process are the same as those used in the detection process.

[0061] (6) Solid-phase extraction purification Methanol activation volume 3.5 mL, flow rate 0.75 mL / min; n-hexane activation volume 3.5 mL, flow rate 0.75 mL / min; sample loading flow rate 0.55 mL / min, sample volume 3 mL; eluent (n-hexane: ethyl acetate = 9:1), volume 5.5 mL, flow rate 0.75 mL / min; eluent (methanol: dichloromethane = 2:1), volume 3.5 mL, flow rate 0.45 mL / min; (7) HPLC detection Mobile phase (methanol:acetonitrile:dichloromethane = 50:40:10); flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 470 nm; injection volume 15 μL.

[0062] The test results of the three concentrations of simulated diets are shown in Table 2.

[0063] Table 2. Detection results of three concentrations of simulated diets

[0064] Example 3 (1) Activation by hydrolysis of silica microspheres Silica microspheres with a particle size of 100 μm and a specific surface area of ​​300 m² 2 / g; hydrochloric acid concentration 1.2mol / L; solid-liquid ratio 1g:15mL; hydrolysis temperature 70℃ (within the range of 68-72℃); reaction time 3h; (2) Surface modification and urea grafting Activated silica microspheres: dichloromethane: triethylamine = 1 g: 22 mL: 3.5 mL; initial modification temperature 30℃, time 2 h; mixed solvent (dichloromethane: anhydrous ethanol = 4: 1); surface-modified silica microspheres: mixed solvent: 4-aminophenylurea = 1: 7.5: 0.55 (mass ratio); grafting temperature 50℃ (within the range of 48-52℃), time 4.5 h; (3) Introduction of antioxidant groups and silanol blockade Urea-based silica microspheres: ascorbate palmitate: organic solvent (methanol) = 1g: 0.2g: 12mL; mixing reaction temperature 60℃, time 3h; trimethylchlorosilane addition amount 7.5% (total mass of reaction system); sealing temperature 30℃, time 1.5h; (4) Sample extraction and preparation of solid phase extraction column The sample (a simulated diet supplemented with astaxanthin) was ground to 60 mesh; the sample-acetonitrile ratio was 1 g: 7 mL (7-8 mL range); the shaking speed was 190 r / min (190-210 r / min range), and the extraction time was 14 min (14-16 min range); the centrifugation speed was 10000 r / min, and the time was 7 min (7-8 min range); the extraction column volume was 5.5 mL; the bonding material packing amount was 0.2 g; the ethanol washing volume was 3.5 mL, the flow rate was 1.5 mL / min; and the nitrogen purging pressure was 0.2 MPa, and the time was 4 min. (5) Drawing the standard curve Prepare astaxanthin standard solutions (methanol as solvent) with a concentration gradient of 0.01-1.0 μg / mL. Use methanol as a blank control to subtract solvent background interference. Plot the concentration of the standard working solution (x, unit: μg / mL) on the x-axis and the corresponding average peak area (y, peak area × 10) on the y-axis. 4 Using AU as the ordinate, a linear regression analysis was performed. The linear regression equation was y = 32.92x - 0.010, and the correlation coefficient R0 was [value missing]. 2 =0.9998, the HPLC parameters used in the standard curve plotting process are the same as those used in the detection process.

[0065] (6) Solid-phase extraction purification Methanol activation volume 3.5 mL, flow rate 0.75 mL / min; n-hexane activation volume 3.5 mL, flow rate 0.75 mL / min; sample loading flow rate 0.55 mL / min, sample volume 3 mL; eluent (n-hexane: ethyl acetate = 9:1), volume 5.5 mL, flow rate 0.75 mL / min; eluent (methanol: dichloromethane = 2:1), volume 3.5 mL, flow rate 0.45 mL / min; (7) HPLC detection Mobile phase (methanol:acetonitrile:dichloromethane = 50:40:10); flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 470 nm; injection volume 15 μL.

[0066] The test results of the three concentrations of simulated diets are shown in Table 3.

[0067] Table 3. Detection results of three concentrations of simulated diets

[0068] Example 4 (1) Activation by hydrolysis of silica microspheres Silica microspheres with a particle size of 100 μm and a specific surface area of ​​300 m² 2 / g; hydrochloric acid concentration 1.2mol / L; solid-liquid ratio 1g:15mL; hydrolysis temperature 70℃ (within the range of 68-72℃); reaction time 3h; (2) Surface modification and urea grafting Activated silica microspheres: dichloromethane: triethylamine = 1 g: 22 mL: 3.5 mL; initial modification temperature 30℃, time 2 h; mixed solvent (dichloromethane: anhydrous ethanol = 4: 1); surface-modified silica microspheres: mixed solvent: 4-aminophenylurea = 1: 7.5: 0.55 (mass ratio); grafting temperature 50℃ (within the range of 48-52℃), time 4.5 h; (3) Introduction of antioxidant groups and silanol blockade Urea-based silica microspheres: ascorbate palmitate: organic solvent (methanol) = 1g: 0.2g: 12mL; mixing reaction temperature 60℃, time 3h; trimethylchlorosilane addition amount 7.5% (total mass of reaction system); sealing temperature 30℃, time 1.5h; (4) Sample extraction and preparation of solid phase extraction column The sample (a simulated diet supplemented with astaxanthin, 0.05 mg / g spiked at a medium concentration) was ground to 60 mesh; sample: acetonitrile = 1 g: 7.5 mL; shaking speed 200 r / min, extraction time 15 min; centrifugation speed 10000 r / min, time 7.5 min; extraction column volume 5.5 mL; 0.2 g of bonding material after being treated with "ethanol washing 3.5 mL (1.5 mL / min) → nitrogen purging 0.2 MPa, 4 min" in Example 1 was loaded; ethanol washing volume 3.5 mL, flow rate 1.5 mL / min; nitrogen purging pressure 0.2 MPa, time 4 min; (5) Drawing the standard curve Prepare astaxanthin standard solutions (methanol as solvent) with a concentration gradient of 0.01-1.0 μg / mL. Use methanol as a blank control to subtract solvent background interference. Plot the concentration of the standard working solution (x, unit: μg / mL) on the x-axis and the corresponding average peak area (y, peak area × 10) on the y-axis. 4 Using AU as the ordinate, a linear regression analysis was performed. The linear regression equation is y = 32.80x - 0.013, and the correlation coefficient R0 is [value missing]. 2 =0.9997, the HPLC parameters used in the standard curve plotting process are the same as those used in the detection process.

[0069] (6) Solid-phase extraction purification Methanol activation volume 3.5 mL, flow rate 0.75 mL / min; n-hexane activation volume 3.5 mL, flow rate 0.75 mL / min; sample loading flow rate 0.55 mL / min, sample volume 3 mL; eluent (n-hexane: ethyl acetate = 9:1), volume 5.5 mL, flow rate 0.75 mL / min; eluent (methanol: dichloromethane = 2:1), volume 3.5 mL, flow rate 0.45 mL / min; (7) HPLC detection Mobile phase (methanol:acetonitrile:dichloromethane = 50:40:10); flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 470 nm; injection volume 15 μL.

[0070] The test results of the three concentrations of simulated diets are shown in Table 4.

[0071] Table 4. Detection results of three concentrations of simulated diets

[0072] Comparative Example 1 (1) Basic conditions for sample pretreatment The sample (a simulated diet supplemented with astaxanthin) was ground to 60 mesh; sample: acetonitrile = 1g: 7.5mL; shaking speed 200r / min, extraction time 15min; centrifugation speed 10000r / min, time 7.5min; (consistent with Example 1) (2) Preparation of solid phase extraction column (replaced with C18 column) A commercially available C18 solid-phase extraction column (5 mL, filled with 0.2 g C18 adsorbent) was used, eliminating the need for homemade bonding materials; pretreatment was performed according to the standard C18 column procedure: activation with 5 mL of methanol → equilibration with 5 mL of ultrapure water, ready for use. (3) Drawing the standard curve Prepare astaxanthin standard solutions (methanol as solvent) with a concentration gradient of 0.01-1.0 μg / mL. Use methanol as a blank control to subtract solvent background interference. Plot the concentration of the standard working solution (x, unit: μg / mL) on the x-axis and the corresponding average peak area (y, peak area × 10) on the y-axis. 4 Using AU as the ordinate, a linear regression analysis was performed. The linear regression equation was y = 32.65x - 0.020, and the correlation coefficient R0 was [value missing]. 2 =0.9995, the HPLC parameters used in the standard curve plotting process are the same as those used in the detection process.

[0073] (4) Solid phase extraction purification (C18 column under normal conditions) The sample loading flow rate was 0.55 mL / min, and the sample loading volume was 3 mL; the eluent was a methanol-water (20:80) mixture, with a volume of 5.5 mL and a flow rate of 0.75 mL / min; the eluent was pure methanol, with a volume of 5 mL and a flow rate of 0.45 mL / min; the eluent was collected. (5) HPLC detection Mobile phase (methanol:acetonitrile:dichloromethane = 50:40:10); flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 470 nm; injection volume 15 μL.

[0074] The test results of the three concentrations of simulated diets are shown in Table 5.

[0075] Table 5. Detection results of three concentrations of simulated diets

[0076] Comparative Example 2 (1) Activation by hydrolysis of silica microspheres Silica microspheres with a particle size of 100 μm and a specific surface area of ​​300 m² 2 / g; hydrochloric acid concentration 1.2mol / L; solid-liquid ratio 1g:15mL; hydrolysis temperature 70℃ (within the range of 68-72℃); reaction time 3h; (2) Surface modification and urea grafting Activated silica microspheres: dichloromethane: triethylamine = 1 g: 22 mL: 3.5 mL; initial modification temperature 30℃, time 2 h; mixed solvent (dichloromethane: anhydrous ethanol = 4: 1); surface-modified silica microspheres: mixed solvent: 4-aminophenylurea = 1: 7.5: 0.55 (mass ratio); grafting temperature 50℃ (within the range of 48-52℃), time 4.5 h; (3) Preparation of bonding materials (omitted antioxidant and blocking steps) Only the urea group grafting was retained, omitting the steps of "ascorbate palmitate introduction" and "trimethylchlorosilane blocking"; after the reaction was completed, the mixture was filtered, washed (methanol → dichloromethane → n-hexane), and vacuum dried (60℃, 4h) to obtain a bonded material containing only urea groups; (4) Sample extraction and preparation of solid phase extraction column The sample (a simulated diet supplemented with astaxanthin) was ground to 60 mesh; the sample-acetonitrile ratio was 1 g: 7.5 mL; the shaking speed was 200 r / min, and the extraction time was 15 min; the centrifugation speed was 10000 r / min, and the time was 7.5 min; the extraction column volume was 5.5 mL; the bonding material packing amount was 0.2 g; the ethanol washing volume was 3.5 mL, and the flow rate was 1.5 mL / min; the nitrogen purging pressure was 0.2 MPa, and the time was 4 min. (5) Drawing the standard curve Prepare astaxanthin standard solutions (methanol as solvent) with a concentration gradient of 0.01-1.0 μg / mL. Use methanol as a blank control to subtract solvent background interference. Plot the concentration of the standard working solution (x, unit: μg / mL) on the x-axis and the corresponding average peak area (y, peak area × 10) on the y-axis. 4 Using AU as the ordinate, a linear regression analysis was performed. The linear regression equation was y = 32.70x - 0.018, and the correlation coefficient R0 was [value missing]. 2 =0.9996, the HPLC parameters used in the standard curve plotting process are the same as those used in the detection process.

[0077] (6) Solid-phase extraction purification Methanol activation volume 3.5 mL, flow rate 0.75 mL / min; n-hexane activation volume 3.5 mL, flow rate 0.75 mL / min; sample loading flow rate 0.55 mL / min, sample volume 3 mL; eluent (n-hexane: ethyl acetate = 9:1), volume 5.5 mL, flow rate 0.75 mL / min; eluent (methanol: dichloromethane = 2:1), volume 3.5 mL, flow rate 0.45 mL / min; (7) HPLC detection Mobile phase (methanol:acetonitrile:dichloromethane = 50:40:10); flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 470 nm; injection volume 15 μL.

[0078] The test results of the three concentrations of simulated diets are shown in Table 6.

[0079] Table 6. Detection results of three concentrations of simulated diets

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A rapid detection method for astaxanthin in broiler diets, characterized in that, Includes the following steps: 1) Activated silica microspheres are obtained by mixing silica microspheres with hydrochloric acid solution and carrying out a hydrolysis reaction; 2) Under anaerobic conditions, activated silica microspheres are mixed with dichloromethane and triethylamine to carry out a preliminary reaction to obtain surface-modified silica microspheres. Then, the surface-modified silica microspheres are mixed with a mixed solvent and 4-aminophenylurea to carry out a grafting reaction to obtain silica microspheres with urea groups on the surface. 3) The silica microspheres with urea groups on the surface are mixed and reacted with ascorbate palmitate and organic solvent to introduce antioxidant groups on the surface of the silica microspheres. Trimethylchlorosilane is added to the reaction system to continue the reaction and block the residual silanol groups to obtain astaxanthin-specific bonding materials. 4) Take a sample of broiler feed, grind it, mix it with acetonitrile, shake and extract, centrifuge and collect the supernatant; The bonding material was loaded into the extraction column, and the extraction column was successively washed with ethanol and purged with nitrogen to obtain a solid-phase extraction column. 5) The solid-phase extraction column was activated sequentially with methanol and n-hexane, and then the sample was loaded, washed, and eluted to obtain the eluent; 6) After drying the eluent with nitrogen, it was reconstituted with methanol and detected by high performance liquid chromatography to calculate the astaxanthin content in the broiler diet.

2. The rapid detection method for astaxanthin in broiler feed according to claim 1, characterized in that, The silica microspheres mentioned in step 1) have a particle size of 50-150 μm and a specific surface area of ​​200-400 m². 2 / g; The concentration of the hydrochloric acid solution is 0.5-2 mol / L.

3. The rapid detection method for astaxanthin in broiler feed according to claim 1, characterized in that, The solid-liquid ratio of the silica microspheres to the hydrochloric acid solution in step 1) is 1g:10-20mL; The hydrolysis reaction temperature is 60-80℃, and the reaction time is 2-4h.

4. The rapid detection method for astaxanthin in broiler feed according to claim 1, characterized in that, In step 2), the ratio of activated silica microspheres, dichloromethane, and triethylamine is 1g: 15-30mL: 2-5mL. In step 2), the reaction temperature for modifying the surface of the silica microspheres is 25-35℃, and the reaction time is 1-3h.

5. The rapid detection method for astaxanthin in broiler feed according to claim 1, characterized in that, The mixed solvent mentioned in step 2) is composed of dichloromethane and anhydrous ethanol in a volume ratio of 3-5:1; The mass ratio of the surface-modified silica microspheres to the mixed solvent and 4-aminophenylurea is 1:5-10:0.3-0.8; The grafting reaction is carried out at a temperature of 40-60℃ for 3-6 hours.

6. The rapid detection method for astaxanthin in broiler diets according to claim 1, characterized in that, In step 3), the mixing ratio of the urea-based silica microspheres on the surface to ascorbate palmitate and organic solvent is 1g:0.1-0.3:10-15mL, the mixing reaction temperature is 50-70℃, and the time is 2-4h. The amount of trimethylchlorosilane added is 5%-10% of the total mass of the reaction system. After adding trimethylchlorosilane, the reaction temperature is controlled at 25-35℃ and the reaction time is 1-2h.

7. The rapid detection method for astaxanthin in broiler feed according to claim 1, characterized in that, The particle size of the broiler diet sample after grinding in step 4) is 40-80 mesh, the mixing ratio of broiler diet sample and acetonitrile is 1g: 5-10mL, the shaking extraction speed is 150-250r / min, and the extraction time is 10-20min. The centrifugation speed is 8000-12000 r / min, and the centrifugation time is 5-10 min; The extraction column has a column volume of 3-6 mL, a bonding material loading of 0.1-0.3 g, an ethanol washing volume of 2-5 mL, a washing flow rate of 1-2 mL / min, a nitrogen purging pressure of 0.1-0.3 MPa, and a purging time of 3-5 min.

8. The rapid detection method for astaxanthin in broiler diets according to claim 1, characterized in that, The activation volume of methanol in step 5) is 2-5 mL, and the activation flow rate is 0.5-1 mL / min; The activation volume of the n-hexane is 2-5 mL, and the activation flow rate is 0.5-1 mL / min.

9. The rapid detection method for astaxanthin in broiler diets according to claim 1, characterized in that, In step 5), the sample loading flow rate is 0.3-0.8 mL / min, and the sample loading volume is 1-5 mL. The rinsing agent used in the rinsing process is a mixture of n-hexane and ethyl acetate, with a volume ratio of n-hexane to ethyl acetate of 8-10:1; The rinsing volume is 3-6 mL, and the rinsing flow rate is 0.5-1 mL / min; The eluent used in the elution process is a mixture of methanol and dichloromethane, with a volume ratio of 1-3:

1. The amount of eluent used in the elution process is 2-5 mL, and the flow rate of the eluent is 0.3-0.6 mL / min.

10. The rapid detection method for astaxanthin in broiler feed according to claim 1, characterized in that, The parameters for high-performance liquid chromatography (HPLC) described in step 6) are as follows: The chromatographic column was a C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The mobile phase is a mixed solution of methanol, acetonitrile, and dichloromethane, with a volume ratio of 40-60:30-50:5-15. The flow rate of the mobile phase is 0.8-1.2 mL / min; The column temperature of the chromatographic column is 25-35℃; The detection wavelength is 470±2nm; The injection volume is 10-20 μL; The astaxanthin content was calculated using the external standard method.

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