阅读说明：本技术 一种测定饲料中斑蝥黄着色剂含量的方法 (Method for measuring content of cantharis yellow colorant in feed ) 是由 章厉劼 孙真峥 王庆红 赵贵 周艺林 郭萍 周泽晓 钱莘莘 孙播东 廖欢 罗永成 于 2019-11-07 设计创作，主要内容包括：本发明公开了一种测定饲料中斑蝥黄着色剂含量的方法,包括：(1)预处理,称取待测饲料样品,加入水、饱和氯化钠、0.05％乙酸溶液、混合提取液涡旋混合后离心,吸取上层萃取液,加入无水乙醇,混匀；在氮气下吹干；用甲基叔丁基醚+乙腈溶液溶解残渣,过滤得供试样品溶液；(2)液相色谱仪分析,测定斑蝥黄着色剂的峰面积；(3)根据前述待测饲料样品的斑蝥黄峰面积,利用斑蝥黄线性回归方程计算待测饲料中斑蝥黄着色剂的质量浓度,再计算待测饲料中斑蝥黄着色剂的含量。本发明方法具有简单、快速、准确、灵敏的特点,适用于饲料中斑蝥着色剂含量的检测分析。(The invention discloses a method for measuring the content of canthaxanthin colorant in feed, which comprises the following steps: (1) pretreating, weighing a feed sample to be detected, adding water, saturated sodium chloride, 0.05% acetic acid solution and mixed extracting solution, carrying out vortex mixing, centrifuging, sucking upper layer extracting solution, adding absolute ethyl alcohol, and mixing uniformly; drying under nitrogen; dissolving the residue with a methyl tert-butyl ether and acetonitrile solution, and filtering to obtain a sample solution; (2) analyzing by a liquid chromatograph, and determining the peak area of the cantharis yellow colorant; (3) and calculating the mass concentration of the canthaxanthin colorant in the feed to be detected by utilizing a canthaxanthin linear regression equation according to the canthaxanthin peak area of the feed sample to be detected, and then calculating the content of the canthaxanthin colorant in the feed to be detected. The method has the characteristics of simplicity, rapidness, accuracy and sensitivity, and is suitable for detecting and analyzing the content of the cantharis colorant in the feed.)

1. A method for measuring the content of canthaxanthin colorant in feed is characterized by comprising the following steps:

(1) pretreatment of

Weighing a feed sample to be detected, adding water, saturated sodium chloride, 0.05% acetic acid solution and mixed extracting solution, mixing in a vortex mode, centrifuging, and sucking upper-layer extracting solution;

repeatedly extracting with mixed extracting solution, wherein the mixed extracting solution is methanol: ethyl acetate: a mixed solution of n-hexane;

mixing the extractive solutions, adding anhydrous ethanol, and mixing; drying under nitrogen;

with methyl tert-butyl ether: dissolving residues in an acetonitrile solution, and filtering to obtain a sample solution;

(2) liquid chromatograph analysis

Analyzing the sample solution to be tested by a liquid chromatograph, and determining the peak area of the canthaxanthin colorant;

(3) calculating content of cantharis yellow colorant in feed

Measuring the cantharis yellow peak area by utilizing a plurality of groups of cantharis yellow solutions with known mass concentrations, and performing linear regression by taking the cantharis yellow mass concentration as a horizontal coordinate and taking a corresponding peak area as a vertical coordinate to establish a cantharis yellow linear regression equation;

and calculating the mass concentration of the canthaxanthin colorant in the feed to be detected by utilizing a canthaxanthin linear regression equation according to the canthaxanthin peak area of the feed sample to be detected, and then calculating the content of the canthaxanthin colorant in the feed to be detected.

2. The method for determining the content of canthaxanthin colorant in feed according to claim 1, wherein the canthaxanthin linear regression equation is:

y＝40401.4x-1776.1

in the formula: y is the cantharis yellow peak area, and x is the mass concentration of the cantharis yellow colorant in the feed to be detected.

3. The method for determining the content of canthaxanthin colorant in feed according to claim 1, wherein the formula for calculating the content of canthaxanthin colorant in feed is:

in the formula, W is the content of the canthaxanthin colorant in the feed to be detected, x is the mass concentration of the canthaxanthin colorant in the feed to be detected, V is the volume of the methyl tert-butyl ether and acetonitrile solution of the dissolved residue, and m is the mass of the feed to be detected.

4. The method for determining the content of canthaxanthin colorant in feed according to claim 1, wherein the ratio of methanol: ethyl acetate: the volume ratio of n-hexane is 1: 1: 1.

5. the method for determining the content of canthaxanthin colorant in feed according to claim 1, wherein the ratio of methyl tert-butyl ether to acetonitrile in the methyl tert-butyl ether + acetonitrile solution is: the volume ratio of acetonitrile is 1: 9.

6. the method for determining the content of canthaxanthin colorant in feed according to claim 1, wherein the detection range of the content of canthaxanthin in the feed is 0.20-25 mg/kg.

Technical Field

The invention relates to a method for measuring the content of canthaxanthin colorant in feed, belonging to the technical field of canthaxanthin colorant content detection.

Background

At present, the highest limit of β -apo-8 ' - β -carotene-4, 4-diketone (canthaxanthin) in the compound feed is clearly specified in No. 2625 bulletin of Ministry of agriculture of the people's republic of China to be 25 mg/kg., the industry standard of β -apo-8 ' - β -carotene-4, 4-diketone (canthaxanthin) detection in the feed in China adopts forward chromatography, the universality of the method is poor, the detection blank of the colorant in the feed is caused, and the disadvantages of quality safety problem and supervision of the feed product are increased.

β -apo-8' - β -carotene-4, 4-dione (canthaxanthin) is also called canthaxanthin, is a carotenoid pigment with orange red, is mostly used as an animal feed additive to make the color of egg yolk and poultry meat more vivid, is mainly used as an orange colorant when being used for food addition, but is rarely used.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the method for determining the content of the canthaxanthin colorant in the feed has high sensitivity, good accuracy and high analysis speed, and solves the problems of interference of lipid and protein in a sample on a detection result and instability of canthaxanthin to be detected in the prior art through reasonable sample extraction and purification conditions.

The technical scheme of the invention is as follows: a method for determining content of canthaxanthin colorant in feed comprises:

(1) pretreatment of

Weighing a feed sample to be detected, adding water, saturated sodium chloride, 0.05% acetic acid solution and mixed extracting solution, mixing in a vortex mode, centrifuging, and sucking upper-layer extracting solution;

repeatedly extracting with mixed extract to obtain upper layer extract, wherein the mixed extract is methanol: ethyl acetate: a mixed solution of n-hexane;

mixing the extractive solutions, adding anhydrous ethanol, and mixing; drying under nitrogen;

with methyl tert-butyl ether: dissolving residues in an acetonitrile solution, and filtering to obtain a sample solution;

(2) liquid chromatograph analysis

Analyzing the sample solution to be tested by a liquid chromatograph, and determining the peak area of the canthaxanthin colorant;

(3) calculating content of cantharis yellow colorant in feed

Measuring the cantharis yellow peak area by utilizing a plurality of groups of cantharis yellow solutions with known mass concentrations, and performing linear regression by taking the cantharis yellow mass concentration as a horizontal coordinate and taking a corresponding peak area as a vertical coordinate to establish a cantharis yellow linear regression equation;

and calculating the mass concentration of the canthaxanthin colorant in the feed to be detected by utilizing a canthaxanthin linear regression equation according to the canthaxanthin peak area of the feed sample to be detected, and then calculating the content of the canthaxanthin colorant in the feed to be detected.

Preferably, the cantharidin linear regression equation is as follows:

y＝40401.4x-1776.1

in the formula: y is the cantharis yellow peak area, and x is the mass concentration of the cantharis yellow colorant in the feed to be detected.

Preferably, the calculation formula of the cantharis yellow colorant content in the feed is as follows:

in the formula, W is the content of the canthaxanthin colorant in the feed to be detected, x is the mass concentration of the canthaxanthin colorant in the feed to be detected, V is the volume of the methyl tert-butyl ether and acetonitrile solution of the dissolved residue, and m is the mass of the feed to be detected.

Preferably, the ratio of methanol: ethyl acetate: the volume ratio of n-hexane is 1: 1: 1.

preferably, the methyl tert-butyl ether: methyl tert-butyl ether in acetonitrile solution: the volume ratio of acetonitrile is 1: 9.

preferably, the detection range of the cantharidin content in the feed is 0.20-25 mg/kg.

The invention has the beneficial effects that: the method takes the colorant canthaxanthin under the colorant item in feed additive safety use standard as a research object, a feed sample to be detected is mixed with water, saturated sodium chloride and 0.05 percent acetic acid solution, starch coated on the surface of the canthaxanthin is dissolved, mixed extract liquid-liquid extraction is carried out by using methanol, ethyl acetate and n-hexane, and after the upper layer solution is concentrated and redissolved, the detection is carried out by using the ultra-high performance liquid chromatography.

The method is beneficial to establishing a content detection standard of canthaxanthin colorants in the feed and providing a large amount of basic data, gradually perfects the basis for monitoring the feed quality safety risk, evaluates the quality safety of canthaxanthin, masters the consumption of canthaxanthin in feed production, and guides feed enterprises and farms to scientifically and reasonably use the colorants, thereby ensuring that no major quality safety incidents of poultry and livestock products occur and reducing the production cost under the condition of meeting the color requirements of consumers.

Drawings

FIG. 1 is a liquid chromatogram of a cantharis yellow standard sample.

Detailed Description

The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.