阅读说明：本技术 一种同时测定鸡蛋中7种色素的方法 (Method for simultaneously determining 7 pigments in eggs ) 是由 杨成 黄晴 沈晓芳 孙嵇成 于 2020-06-08 设计创作，主要内容包括：本发明公开了一种同时测定鸡蛋中7种色素的方法,属于食品安全检测技术领域。所述方法是采用DQTP/Fe<Sub>3</Sub>O<Sub>4</Sub>有机共价骨架结构和磁性纳米复合材料,作为分散式磁性固相萃取吸附剂,以提取鸡蛋中的色素,再采用超高效液相色谱串联质谱UPLC-MS/MS法结合外标法测定鸡蛋中色素含量。本发明建立一种同时测定鸡蛋中核黄素、叶黄素、角黄素和苏丹红Ⅰ、苏丹红Ⅱ、苏丹红Ⅲ、苏丹红Ⅳ色素含量方法。叶黄素、角黄素、苏丹红Ⅰ、苏丹红Ⅱ、苏丹红Ⅲ、苏丹红Ⅳ的检出限均为0.2ug/kg,核黄素的检出限为0.2ug/kg。7种色素在加标量2～10ug/kg范围内,回收率为80.2～106.7％,相对标准偏差RSD为1.5～6.0％。(The invention discloses a method for simultaneously determining 7 pigments in eggs, and belongs to the technical field of food safety detection. The method adopts DQTP/Fe 3 O 4 Organic covalent skeleton structure and magnetic nano composite material as dispersive magnetic solid phase extractionAnd (3) taking an adsorbent to extract the pigment in the eggs, and determining the content of the pigment in the eggs by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry UPLC-MS/MS method in combination with an external standard method. The invention provides a method for simultaneously measuring the contents of riboflavin, lutein, canthaxanthin, Sudan red I, Sudan red II, Sudan red III and Sudan red IV pigments in eggs. The detection limits of lutein, canthaxanthin, Sudan red I, Sudan red II, Sudan red III and Sudan red IV are all 0.2ug/kg, and the detection limit of riboflavin is 0.2 ug/kg. The recovery rate of the 7 pigments is 80.2-106.7% within the range of adding standard amount of 2-10 ug/kg, and the relative standard deviation RSD is 1.5-6.0%.)

1. A pretreatment method for extracting pigment from eggs is characterized in that DQTP/Fe is adopted₃O₄The magnetic nano material is used as a dispersed magnetic solid phase extraction adsorbent to extract pigment in the eggs; the DQTP/Fe₃O₄The magnetic nano material is prepared by adopting DQTP to Fe₃O₄The magnetic nano material is prepared by surface modification.

2. The pretreatment method according to claim 1, wherein the DQTP/Fe is prepared by coprecipitation₃O₄A magnetic nanomaterial; the reaction conditions are as follows: heating in water bath; DQTP and Fe₃O₄The mass ratio of the magnetic nano material is (1-5): (1-2); the temperatures are respectively 80-90 ℃.

3. The pretreatment method according to claim 1 or 2, wherein the pretreatment method is DQTP/Fe₃O₄Adding magnetic nanometer material into sample to be tested, adsorbing for 15-30min, and adsorbing DQTP/Fe with magnet₃O₄A magnetic nanomaterial; eluting DQTP/Fe with methanol₃O₄Magnetic nano materialThe pigment of (1).

4. The pretreatment method according to any one of claims 1 to 3, wherein the pigment comprises one or more of lutein, riboflavin, canthaxanthin, Sudan I, Sudan II, Sudan III and Sudan IV.

5. A method for detecting the pigment content in eggs is characterized in that the method comprises the steps of treating egg samples by the pretreatment method of any one of claims 1 to 4, and then measuring the pigment content in the eggs by an ultra performance liquid chromatography-tandem mass spectrometry UPLC-MS/MS method in combination with an external standard method.

6. The method of claim 5, wherein the column of the ultra high performance liquid chromatography is a 2.6 μm, 100 x 3.0mm phenomenex kinetexf5 column.

7. The method according to claim 5 or 6, wherein the mobile phase of the ultra-high performance liquid chromatography is: mobile phase A: 0.1% by volume of formic acid/water solution; mobile phase B: 0.1% formic acid/acetonitrile solution; the elution conditions were: 0-2min, 20% A + 80% B; 2-4.5min, 100% B; 4.6-6.6min 20% A + 80% B.

8. The method of any of claims 5-7, wherein the mass spectrometry conditions are: an electrospray ion source and a positive ion scanning mode are adopted; the ion source temperature is 550 ℃; curtain gas 30 psi; capillary voltage 5500V; atomization gas pressure 55 psi; the assist gas pressure was 55 psi.

9. Use of the method according to any one of claims 5 to 8 for quality testing of egg products.

10. DQTP/Fe₃O₄The application of the magnetic nano material in the aspect of separating or extracting pigments is characterized in that the DQTP/Fe₃O₄The magnetic nano material is prepared by adopting DQTP to Fe₃O₄The magnetic nano material is prepared by surface modification.

Technical Field

The invention relates to a method for simultaneously determining 7 pigments in eggs, belonging to the technical field of food safety detection.

Background

With the increasing living standard, the pursuit of natural organic food materials by people is becoming a trend. Eggs laid by free-range chickens under natural conditions, commonly called 'native eggs' or 'grass eggs', are favored, and the price is 1 time or even several times higher than that of common eggs. The common consumers distinguish the native eggs from the common eggs mainly by the color of the yolk, and consider the native eggs to be darker. The color of the egg yolk is derived from pigment substances such as riboflavin and lutein. The free-range chickens have more chances to eat foods such as leaves, seeds and the like rich in lutein, wherein the lutein is a natural carotenoid substance and is enriched in egg yolk after being metabolized, so that the egg yolk is bright orange. The cage-raised chickens mainly eat artificial feed, the lutein intake is low, the egg laying period is short, and the egg yolks are mostly light yellow. In order to meet the preference of consumers on the color of the yolk, a plurality of farmers add pigments such as Calligein red, Sudan red and the like into the feed, so that the color of the yellow of the cage-bred eggs is darkened, and the cage-bred eggs are sold at high price as native eggs. Calicidin red has canthaxanthin as main component, is a kind of artificially synthesized carotenoid, and exists in nature only in small amount. Canthaxanthin has little nutritional value, and excessive intake of canthaxanthin may also affect the eyesight of a human. Although Callicarin red is allowed to be added to animal feed, its use in making fake egg is commercially fraudulent and severely impairs consumer awareness. Sudan belongs to azo series dyes, is not a food additive, and most of the dyes have carcinogenicity.

China is a big country for producing and consuming eggs, and the quality safety of eggs is related to the livelihood and affects the market stability. Various endogenous and exogenous pigments in eggs can reflect the quality and safety risks of eggs to a certain extent. A large amount of researches are carried out on the detection of pigment substances in eggs at home and abroad, for example, the HPLC-MS/MS detection method of carotenoid colorants in egg yolks is established by Jiaogui et al, the Sudan red detection method in egg eggs is established by Liu et al, and the high performance liquid chromatography method for detecting canthaxanthin and astaxanthin in egg yolks is established by He Kangha et al. The method has the limitation that the detected target object is single or less, and qualitative and quantitative analysis on various pigment substances in the egg yolk cannot be simultaneously carried out, so that natural nutrient substances and artificially added pigments can be distinguished.

Disclosure of Invention

Aiming at the problems, a magnetic covalent organic framework compound is used as a dispersed solid-phase extracting agent, the absorbing and extracting capabilities of 7 pigments in eggs, namely lutein, riboflavin, canthaxanthin, Sudan red I, Sudan red II, Sudan red III and Sudan red IV are good, and a method for simultaneously detecting the 7 pigments in the eggs is established by a high performance liquid chromatography triple four-level tandem mass spectrometry. The effectiveness and the accuracy of the method are verified by optimizing the detection conditions and detecting the actual sample. Compared with the method reported in the related literature, the method is simple to operate, has strong specificity, has important meaning for detecting the pigment in the eggs, and provides a new idea for detecting the pigment.

The dispersed magnetic solid phase extraction is carried out by using magnetic material or magnetic substance (such as Fe)₃O₄、Fe₂O₃) The modified material is used as an extraction adsorbent, the magnetic adsorbent is directly added into a sample solution containing a target analyte, and the adsorbent is separated from a sample matrix under the action of an external magnetic field after extraction and adsorption. The method is an improvement and promotion on the basis of the dispersed solid-phase extraction, has all the advantages of the dispersed solid-phase extraction, avoids the procedures of centrifugation or filtration, and is more convenient to operate. The magnetic adsorbent is used as the core and key of dispersive magnetic solid phase extraction, and is also the premise for applying the technology.

Fe₃O₄And Fe₂O₃The magnetic nano-particles have small adsorption quantity and no functional groups. The composite magnetic nano material provides a wide application space for dispersed magnetic solid-phase extraction. Covalent Organic Frameworks (COFs) are two-or three-dimensional crystalline porous structures composed of strong covalent bonds between organic subunits. COFs have many unique properties, such as tunable porosity, ultra-large specific surface area, and good chemical and thermal stability. The patent magnetizes COF (DQTP) to prepare magnetic COF nano material (DQTP/Fe)₃O₄) The method is used as a dispersive magnetic solid phase extraction adsorbent and is applied to a sample pretreatment method for detecting 7 pigments in eggs.

The first purpose of the invention is to provide a pretreatment method for extracting pigment from eggs, which adopts DQTP/Fe₃O₄The magnetic nano material is used as a dispersed magnetic solid phase extraction adsorbent to extract pigment in the eggs; the DQTP/Fe₃O₄The magnetic nano material is prepared by adopting DQTP to Fe₃O₄The magnetic nano material is prepared by surface modification. Wherein, DQTP is prepared from 2, 6-Diaminoanthraquinone (DQ) and 1,3, 5-Trimethyl Phloroglucinol (TP).

In one embodiment of the invention, the DQTP/Fe is prepared by adopting a coprecipitation method₃O₄A magnetic nanomaterial; the reaction conditions are as follows: heating in water bath; DQTP and Fe₃O₄The mass ratio of the magnetic nano material is (1-5): (1-2); the temperatures are respectively 80-90 ℃.

In one embodiment of the invention, the pretreatment method is to add DQTP/Fe₃O₄Adding magnetic nanometer material into sample to be detected, adsorbing for 15-30min, and adsorbing DQTP/Fe with magnet₃O₄A magnetic nanomaterial; eluting DQTP/Fe with methanol₃O₄Pigment on the magnetic nanomaterial.

In one embodiment of the invention, the pigment comprises one or more of lutein, riboflavin, canthaxanthin, sudan red i, sudan red ii, sudan red iii and sudan red iv.

The second purpose of the invention is to provide a method for detecting the pigment content in eggs, wherein the method comprises the steps of treating egg samples by adopting the pretreatment method, and then measuring the pigment content in the eggs by adopting an ultra performance liquid chromatography-tandem mass spectrometry UPLC-MS/MS method in combination with an external standard method.

In one embodiment of the invention, the column model of the ultra high performance liquid chromatography is Phenomenex KinetexF5(2.6 μm, 100X 3.0 mm).

In one embodiment of the invention, the mobile phase of the ultra-high performance liquid chromatography is: mobile phase A: 0.1% by volume (v/v) formic acid/water solution; mobile phase B: 0.1% (v/v) formic acid/acetonitrile solution; the elution conditions were: 0-2min, 20% A + 80% B; 2-4.5min, 100% B; 4.6-6.6min 20% A + 80% B.

In one embodiment of the present invention, the mass spectrometry conditions are: an electrospray ion source and a positive ion scanning mode are adopted; the temperature of an ion source is 550 ℃; curtain gas 30 psi; capillary voltage 5500V; atomization gas pressure 55 psi; the assist gas pressures were all 55 psi.

The third purpose of the invention is to provide the application of the method in the quality detection of the egg products.

The fourth purpose of the invention is to provide a DQTP/Fe₃O₄The application of the magnetic nano material in the aspect of separating or extracting pigments; the DQTP/Fe₃O₄The magnetic nano material is prepared by adopting DQTP to Fe₃O₄The magnetic nano material is prepared by surface modification.

The invention has the beneficial effects that:

the invention provides an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneously determining the contents of riboflavin, lutein, canthaxanthin, Sudan red I, Sudan red II, Sudan red III and Sudan red IV pigments in eggs. The 7 pigments have good linear relation in the concentration range of 1-20 ug/L and the correlation coefficient r²Not less than 0.998. The detection limits of lutein, canthaxanthin, Sudan red I, Sudan red II, Sudan red III and Sudan red IV are all 0.2ug/kg, and the detection limit of riboflavin is 0.2 ug/kg. The recovery rate of the 7 pigments is 80.2-106.7% within the range of adding standard amount of 2-10 ug/kg, and the Relative Standard Deviation (RSD) is 1.5-6.0% of the total weight of the composition. The method is accurate, rapid (the total time of pretreatment and detection is only 40min), and efficient, and can be used for quantitative and quantitative analysis of internal and external pigments in eggs.

Drawings

FIG. 1 is an extracted ion chromatogram of 7 pigments.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.