阅读说明：本技术 食品中胭脂虫红含量的高效液相色谱检测方法 (High performance liquid chromatography detection method for content of cochineal in food ) 是由 谢亚楠 李悦 俞涵 鲁瑶 尹亮 朱敬华 郭兴旺 于 2019-12-23 设计创作，主要内容包括：本发明涉及一种食品中胭脂虫红含量的高效液相色谱检测方法,其特征在于：该方法包括如下步骤：1)标准工作液的配制；2)样品工作液的配制：3)高效液相色谱法检测分析。本发明设计科学合理,能够对食品中的胭脂虫红含量进行准确的分析检测,使得分析前处理过程更简单快捷,成分检测分析更快速准确,节省时间,且检测灵敏度较高,检测数据准确,适用范围广,节约成本,提高检测效率。(The invention relates to a high performance liquid chromatography detection method for cochineal content in food, which is characterized by comprising the following steps: the method comprises the following steps: 1) preparing a standard working solution; 2) preparing a sample working solution: 3) and (4) detecting and analyzing by using a high performance liquid chromatography. The method is scientific and reasonable in design, can be used for accurately analyzing and detecting the content of the cochineal in the food, so that the pretreatment process of analysis is simpler and quicker, the component detection and analysis are quicker and more accurate, the time is saved, the detection sensitivity is higher, the detection data is accurate, the application range is wide, the cost is saved, and the detection efficiency is improved.)

1. A high performance liquid chromatography detection method for cochineal content in food is characterized in that: the method comprises the following steps:

1) preparing a standard working solution:

accurately weighing a proper amount of carminic acid standard substance, dissolving and diluting the carminic acid standard substance by using methanol, transferring the carminic acid standard substance to a volumetric flask, and fixing the volume to prepare carminic acid standard working solution with proper concentration for later use;

2) preparing a sample working solution:

accurately weighing 0.3-0.5 g of sample in a 50mL centrifuge tube, adding 10mL of 2mol/L HCl extracting solution, carrying out vortex, carrying out ultrasonic treatment at 75 ℃ for 30min, cooling to room temperature, adding 15mL of methanol, oscillating, cooling to room temperature, centrifuging at 8000rpm for 3min, taking supernatant, carrying out reduced pressure evaporation at 45 ℃ to about 7mL, passing through a solid phase extraction small column, collecting effluent, carrying out reduced pressure evaporation in a water bath at 45 ℃ until the effluent is nearly dry, re-dissolving with 1mL of mobile phase, and carrying out HPLC analysis after passing through a membrane;

3) high performance liquid chromatography detection and analysis:

respectively injecting the standard working solution and the sample working solution into a high performance liquid chromatograph, performing high performance liquid chromatography analysis, quantitatively comparing the peak area of the standard working solution with the peak area of the sample working solution, determining the retention time, performing calculation by an external standard method, and calculating the content of cochineal in the sample.

2. The high performance liquid chromatography detection method for the content of cochineal in food according to claim 1, wherein the detection method comprises: the content of the cochineal in the step 3) is calculated by adopting the following formula:

X_i＝C×V×1000/m×1000

wherein: xi is the content of cochineal in the substance to be detected in the sample, and the unit is mg/kg;

c is the concentration of a substance to be detected in the determination solution, and the unit is mu g/mL;

v is the reconstitution volume, and the unit is mL;

m is the sample size in g;

1000-unit conversion factor.

3. The high performance liquid chromatography detection method for the content of cochineal in food according to claim 1, wherein the detection method comprises: the liquid chromatogram conditions are as follows:

a chromatographic column: RP-C₁₈5μm 150mm×4.6mm；

Mobile phase: a is 0.1 percent formic acid solution, B is acetonitrile, and equal gradient elution is adopted;

flow rate: 0.4-1.5 mL/min;

column temperature: 25-40 ℃;

sample introduction amount: 2-20 μ L.

4. The high performance liquid chromatography detection method for the content of cochineal in food according to claim 3, wherein the detection method comprises: in the liquid chromatogram, the proportion of the mobile phase is A: b15: 85.

Technical Field

The invention belongs to the field of food additive detection, and relates to a high performance liquid chromatography detection method for cochineal content in food.

Background

Cochineal red is extracted from cochineal grown on Opuntia ficus-indica, is an anthraquinone natural pigment, is different from other natural pigments, has stable physicochemical properties, and is regarded as the safest natural pigment for production of food, cosmetics, medicines, textiles, etc. At present, China allows the production of the food additive. Because cochineal is a natural pigment with natural and bright color, and has great advantages over synthetic pigments, some large food manufacturers have begun to replace synthetic pigments with cochineal. Cochineal red as a natural pigment additive can be used in beverages, meat products, candies, appetizing wine, cider vinegar, yoghurt, bakery products, jam, jelly and the like, and is listed in GB2760-2014 national food safety standard for use of food additives, but the quality standard and the inspection method are not specified. At present, China does not have the standard of a cochineal detection method, but the phenomena of overuse and overlimit addition of the cochineal exist. In order to ensure the quality safety of the fruit juice beverage, it is necessary to develop a method for detecting cochineal in the beverage as soon as possible. Therefore, a detection method for rapidly determining the content of cochineal in the juice beverage by a High Performance Liquid Chromatography (HPLC) method is established, and the method has important supervision significance for quality safety of the juice beverage.

By searching for the published patent documents, no published patent document of a method for detecting the content of cochineal in food by high performance liquid chromatography is found, and no published patent document similar to that of the present patent application is found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art aiming at the natural edible pigment-cochineal red with increasing demands in the current market and aiming at different types of foods added with cochineal red, and provides a high performance liquid chromatography detection method for detecting the cochineal red content in the foods, which has the advantages of simple detection operation, high detection precision, good repeatability, accuracy and high efficiency.

The technical problem to be solved by the invention is realized by the following technical scheme:

a high performance liquid chromatography detection method for content of cochineal in food is characterized in that: the method comprises the following steps:

1) preparing a standard working solution:

weighing a proper amount of carminic acid standard substance, dissolving the carminic acid standard substance by using methanol, transferring the carminic acid standard substance to a volumetric flask, and fixing the volume to prepare a carminic acid standard working solution with a proper concentration for later use;

2) preparing a sample working solution:

accurately weighing 0.3-0.5 g of sample in a 50mL centrifuge tube, adding 10mL of 2mol/L HCl extracting solution, carrying out vortex, carrying out ultrasonic treatment at 75 ℃ for 30min, cooling to room temperature, adding 15mL of methanol, oscillating, cooling to room temperature, centrifuging at 8000rpm for 3min, taking supernatant, carrying out reduced pressure evaporation at 45 ℃ to about 7mL, passing through a solid phase extraction small column, collecting effluent, carrying out reduced pressure evaporation in a water bath at 45 ℃ until the effluent is nearly dry, re-dissolving with 1mL of mobile phase, and carrying out HPLC analysis after passing through a membrane;

3) high performance liquid chromatography detection and analysis:

respectively injecting the standard working solution and the sample working solution into a high performance liquid chromatograph, carrying out chromatographic analysis, carrying out quantitative comparison on the peak area of the standard working solution and the peak area of the sample working solution, carrying out qualitative retention time, carrying out quantitative calculation by an external standard method, and calculating the content of cochineal in the sample.

Moreover, the content of cochineal in the step 3) is calculated by adopting the following formula:

X_i＝(C×V×1000)/(m×1000)

wherein: xi is the content of cochineal in the substance to be detected in the sample, and the unit is mg/kg;

c is the concentration of a substance to be detected in the determination solution, and the unit is mu g/mL;

v is the reconstitution volume, and the unit is mL;

m is the sample size in g;

1000-unit conversion factor.

Moreover, the liquid chromatography conditions are as follows:

a chromatographic column: RP-C₁₈5μm 150mm×4.6mm；

Mobile phase: a is 0.1 percent formic acid solution, B is acetonitrile, and equal gradient elution is adopted;

flow rate: 0.4-1.5 mL/min;

column temperature: 25-40 ℃;

sample introduction amount: 2-20 μ L.

In the liquid chromatography, the ratio of the mobile phase is A: b15: 85.

the invention has the advantages and beneficial effects that:

1. the high performance liquid chromatography detection method for the content of the cochineal in the food is different from most detection methods for the cochineal in the current market, and the analysis method adopting the high performance liquid chromatography can be more targeted and can be used for more quickly and effectively carrying out accurate analysis and detection on the content of the cochineal in the food, so that the component detection and analysis are quicker, and the time is saved.

2. According to the high performance liquid chromatography detection method for the content of the cochineal in the food, the obtained cochineal is good in peak shape and separation degree and good in linearity, can be effectively separated from other impurities in the matrix, avoids interference of other substances, shortens detection time, and is more accurate in detection and extraction.

3. The high performance liquid chromatography detection method for detecting the content of cochineal in food has the advantages of simple pretreatment method process, high extraction efficiency, good purification effect, high detection precision, sensitive detection, short detection time and high detection efficiency.

Drawings

FIG. 1 is a high performance liquid chromatogram of a standard working solution and a sample working solution according to the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.

A high performance liquid chromatography detection method for content of cochineal in food is characterized in that: the method comprises the following steps:

1) preparing a standard working solution:

weighing a proper amount of carminic acid standard substance, dissolving the carminic acid standard substance by using methanol, transferring the carminic acid standard substance to a volumetric flask, and fixing the volume to prepare a carminic acid standard working solution with a proper concentration for later use;

2) preparing a sample working solution:

accurately weighing 0.3-0.5 g of sample in a 50mL centrifuge tube, adding 10mL of 2mol/L HCl extracting solution, carrying out vortex, carrying out ultrasonic treatment at 75 ℃ for 30min, cooling to room temperature, adding 15mL of methanol, oscillating, cooling to room temperature, centrifuging at 8000rpm for 3min, taking supernatant, carrying out reduced pressure evaporation at 45 ℃ to about 7mL, passing through a solid phase extraction small column, collecting effluent, carrying out reduced pressure evaporation in a water bath at 45 ℃ until the effluent is nearly dry, re-dissolving with 1mL of mobile phase, and carrying out HPLC analysis after passing through a membrane;

3) high performance liquid chromatography detection and analysis:

respectively injecting the standard working solution and the sample working solution into a high performance liquid chromatograph, carrying out chromatographic analysis, carrying out quantitative comparison on the peak area of the standard working solution and the peak area of the sample working solution, carrying out qualitative retention time, carrying out quantitative calculation by an external standard method, and calculating the content of cochineal in the sample.

The content of cochineal in step 3) is calculated by adopting the following formula:

X_i＝(C×V×1000)/(m×1000)

wherein: xi-the content of cochineal in the substance to be detected in the sample, wherein the unit is mg/kg;

c-the concentration of the substance to be detected in the determination solution is mu g/mL;

v-reconstitution volume, unit is mL;

m-sample size in g;

1000-unit conversion factor.

The liquid chromatography conditions were:

a chromatographic column: RP-C185 μm 150mm × 4.6 mm;

mobile phase: a is 0.1% formic acid solution, B is acetonitrile, use the equal gradient to elute, the proportion of the mobile phase is A: b15: 85 parts by weight;

flow rate: 0.4-1.5 mL/min;

column temperature: 25-40 ℃;

sample introduction amount: 2-20 μ L.

Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.