CN116930386B - A method for identifying artificially fed royal jelly based on daidzein content - Google Patents

Abstract

The present invention provides a method for identifying artificially fed royal jelly based on daidzein content. Acetonitrile is used to extract daidzein in royal jelly samples, and liquid chromatography tandem mass spectrometry is used to conduct qualitative and quantitative analysis. According to different feeding methods, The difference in daidzein content in the royal jelly produced by bees can be used to identify whether bees are fed feed containing soybean meal. The present invention can identify artificially fed royal jelly based on the content of daidzein. This method has the advantages of being simple, fast, accurate and stable. It supplements the existing identification methods and makes the identification results more convincing. The invention provides a new method for identifying feeding royal jelly, which is of great significance for relevant departments to supervise the quality of royal jelly products, and also facilitates consumers to choose appropriate products according to their own preferences.

Description

A method for identifying artificially fed royal jelly based on daidzein content

Technical field

The invention belongs to the technical field of food and medicine testing, and specifically relates to a method for identifying artificially fed royal jelly based on daidzein content.

Background technique

Royal jelly, also known as royal jelly, is a slurry-like substance secreted by the hypopharyngeal and maxillary glands of young worker bees on the 5th to 14th day after birth. It is the food of the larvae of worker bees and the queen bee throughout their lives. The lifespan of worker bees who consume royal jelly for a short period of time is about It is 35 days, and the life span of a queen bee who consumes royal jelly throughout her life can reach 1-5 years. The water content in royal jelly is about 60%-70%, the crude protein content is about 12%-15%, the total sugar content is about 10%-16%, and the lipid content is about 3%-6%. In addition, it also contains vitamins, Salt, mature amino acids and other substances. Royal jelly has a variety of biological activities and is a natural food suitable for human health. A large number of studies have shown that royal jelly has immunomodulatory effects and helps delay natural immune aging. The unique fatty acid 10-hydroxy-2-decenoic acid (10-HDA) in royal jelly has a potential role in the treatment of immunocompromised cells and can effectively enhance antigens. Specific immune response. In addition, royal jelly also has strong antibacterial activity and anti-aging effects. At the same time, royal jelly treatment can significantly improve glucose homeostasis disorders and inflammatory responses caused by high-fat diet, and has anti-diabetic and anti-obesity activities.

Bees suck nectar and pollen to produce royal jelly. When external climate conditions or honey powder source conditions are unfavorable, beekeepers usually supplement artificial feed to provide nutrition to the bee colonies, maintain bee product production, and reduce feeding costs. Commonly used artificial feeds include sugar feeds that provide carbohydrate sources, such as sucrose, and protein feeds that provide protein sources, such as soybean meal, plant protein, yeast extract, etc. The international standard "Royal Jelly" issued by the International Standards Organization (ISO) divides royal jelly produced by different feeding methods into two categories: Type 1 bees only eat natural food (pollen, nectar and honey); Type 2 bees eat natural food. and other nutrients (protein, carbohydrate, etc.) and pointed out the differences in the δ ¹³ C value (C13/C12 isotope ratio), sucrose, pyranosylsucrose, maltose and maltotriose indicators of the two types of royal jelly, and made Classification requirements. In existing research reports, the methods for identifying royal jelly produced by different feeding methods are mainly based on analyzing the differences in sugar content and δ ¹³ C value in royal jelly. Wytrychowski et al. found that the sugar content and δ ¹³ C value of royal jelly produced based on sugar cane or corn starch hydrolyzate feeding were significantly different from those of non-fed royal jelly. It was also found that regardless of whether protein feed was fed, the δ ¹³ C value of royal jelly produced when honey was fed sucrose or corn hydrolyzate was as high as -17‰, and the contents of maltose, maltotriose, sucrose and glucopyranosyl sucrose also increased significantly.

The method for identifying fed royal jelly in existing research reports is mainly by measuring the various sugar contents and δ ¹³ C differences in royal jelly. Research by Wytrychowski et al. shows that the sucrose and pyranosyl groups of royal jelly fed with exogenous sugars The contents of sucrose, maltose and maltotriose and the δ ¹³ C value were higher than those of naturally fed royal jelly. Among them, the sucrose content of royal jelly obtained by feeding sugar cane or sugar beet can reach up to 7.7%, and the sucrose content of pyranosyl sucrose is as high as 1.7%, which is three times higher than the content in royal jelly fed naturally; the content of royal jelly obtained by feeding cereals and corn starch hydrolyzate The maltose content of royal jelly is 1.4%-5.5%, which is 2-5 times that of naturally fed royal jelly; the maltotriose content is 0.3%-1.7%, which is 2-8 times that of naturally fed royal jelly; fed with sucrose or corn hydrolyzate The δ ¹³ C value of the obtained royal jelly is as high as -17‰, while the δ ¹³ C value of naturally fed royal jelly is -26.45~-23.73%. However, the study found that the difference in the above components mainly comes from feeding sugar feed, while feeding protein feed has no significant impact on the above measurement results. In the production process of royal jelly, sufficient protein source supply is indispensable (natural protein source: bee pollen, protein feed: soybean meal, etc.). The existing methods are difficult to identify the royal jelly produced by feeding artificial protein feed.

Because soybean meal is cheap and easy to obtain, soybean meal is the main raw material for bee artificial protein feed in the Chinese market. Therefore, using artificial protein feeds, especially the influence of small molecule specific components in soybean meal on royal jelly components, to establish the identification of royal jelly in artificial protein feeds can supplement existing identification methods.

Contents of the invention

The purpose of the present invention is to provide a method for identifying artificially fed royal jelly based on daidzein content.

In order to achieve the purpose of the present invention, the present invention provides a method for identifying artificially fed royal jelly based on daidzein content. Acetonitrile is used to extract daidzein in royal jelly samples, and then liquid chromatography-tandem mass spectrometry is used to conduct qualitative and quantitative analysis. , based on the difference in daidzein content in the royal jelly produced by bees under different feeding methods, we can identify whether bees are fed soybean meal feed.

Further, the method includes the following steps:

S1. Standard solution preparation: Use methanol to prepare the following series of daidzein standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg/L, 10 µg/L;

S2. Preparation of test solution: Weigh 2g of royal jelly into a 50 mL centrifuge tube, add 5 mL of zinc acetate buffer solution (ρ=219g/L), vortex for 1-3 min, add 20 mL of acetonitrile, vortex for 1-3 min, and then Add 1g NaCl and 4g Na ₂ SO ₄ , vortex for 1-3 minutes, and centrifuge at 8000r/min for 5-10 minutes at 4°C; take 8mL of the supernatant and add 400 mg of PSA (N-propylethylenediamine), 400 mg C18EC and 200 mg MgSO ₄ in a 15 mL centrifuge tube, vortex for 1-3 min, and centrifuge at 8000 r/min for 5-10 min at 4°C; put 2 mL of the supernatant into a 5 mL centrifuge tube, blow nitrogen until dry, and add 1 Re-dissolve mL of 50% (v/v) methanol solution, vortex for 1-3 minutes, ultrasonic for 1-3 minutes, pass through a 0.2 µm filter membrane to obtain the test solution;

S3. Liquid chromatography tandem mass spectrometry detection: Inject a series of daidzein standard working solutions and test solution respectively, and draw a standard curve with the chromatographic peak area as the ordinate and the concentration of the series of daidzein standard working solutions as the abscissa. , use the standard curve to quantify the sample.

The detection conditions of liquid chromatography tandem mass spectrometry are:

Liquid chromatography conditions are as follows: mobile phase A is 0.1v/v% formic acid aqueous solution, mobile phase B is acetonitrile; elution conditions are shown in Table 1; Agilent Poroshell 120 EC-C18 chromatographic column, 2.1 mm×100 mm, 2.7 µm, column The temperature is 30°C; the flow rate is 0.2 mL/min; the injection volume is 5.0 µL.

Table 1 Liquid chromatography gradient elution table

The mass spectrometry conditions are as follows: electrospray ion source (ESI); positive ion scanning mode; multiple reaction monitoring (MRM) mode; nebulizer temperature 200°C, nebulizer flow rate 15 L/min, nebulizer pressure 30 psi, capillary voltage 3500 V, sheath gas temperature 350°C, sheath gas flow rate 11 L/min.

According to the aforementioned method, the parent ion of daidzein detected by mass spectrometry is 255.0, the quantitative ion is 136.9, and the qualitative ion is 65.2.

Using the aforementioned method, the recovery rate of daidzein is 87.95-117.99%, the detection limit is 0.07µg/kg, and the quantification limit is 0.23µg/kg.

Through the above technical solutions, the present invention has at least the following advantages and beneficial effects:

(1) The present invention uses acetonitrile solution to extract flavonoids from royal jelly samples, and uses liquid chromatography tandem mass spectrometry to conduct qualitative and quantitative analysis. The liquid chromatography tandem mass spectrometry technology used in the present invention uses liquid chromatography as a separation system and mass spectrometry as a detection system. The extracted and purified sample is separated and ionized in the liquid chromatography and mass spectrometry parts, and is obtained through the detector. Mass spectrum. LC-MS embodies the complementary advantages of chromatography and mass spectrometry, combining the high separation ability of chromatography for complex samples with the high selectivity, high sensitivity and ability to provide relative molecular weight and structural information of mass spectrometry.

(2) The present invention can identify feeding royal jelly based on the content of daidzein. The advantages of this method are simple, fast, accurate and stable. It supplements the existing identification methods and makes the identification results more convincing.

(3) The present invention optimizes the extraction reagents for royal jelly. The results show that the highest extraction efficiency can be obtained only by selecting acetonitrile as the extraction solvent and using a C18 purification package to fully remove impurities.

(4) The royal jelly sample solution needs to be diluted with methanol and water before analysis by liquid chromatography tandem mass spectrometer, otherwise the chromatogram will be asymmetric and lead to inaccurate quantification.

(5) 0.1% formic acid needs to be added to the mobile phase of chromatographic detection to provide an acidic environment for the analyte, provide protons, and improve ionization efficiency.

(6) During the analysis of the royal jelly sample solution, only two product ions are contained, and the difference in relative abundance is not more than 10%. Only when the relative deviation of the retention time is no more than 1% can it be determined that the test substance daidzein is contained.

(7) The present invention provides a new method for identifying royal jelly for feeding, which is of great significance for relevant departments to supervise the quality of royal jelly products. It also facilitates consumers to choose appropriate products according to their own preferences.

Description of drawings

Figure 1 shows the structural formula of daidzein.

Figures 2A to 2D are characteristic ion mass chromatograms (MRM) of daidzein in royal jelly measured by liquid chromatography tandem mass spectrometry in the preferred embodiment of the present invention. Among them, Figure 2A shows the relative abundance ratio of qualitative ion/quantitative ion; Figure 2B shows the characteristic ion mass chromatogram (MRM) of daidzein standard solution (2 μg/L); Figure 2C shows the mass chromatogram of quantitative ion (255→136.9) Figure; Figure 2D shows the qualitative ion (255→65.2) mass chromatogram.

Figure 3 shows the content of royal jelly daidzein in different feeding methods in the preferred embodiment of the present invention. ****: P<0.0001 extremely significant level.

Detailed ways

The present invention provides a method for identifying artificially fed royal jelly based on daidzein content. The acetonitrile solution is used to extract soybean isoflavones from royal jelly samples, and then qualitative and quantitative analysis is performed on them. In the royal jelly produced according to different feeding methods, The difference in daidzein content can be used to identify whether soybean meal feed is fed. This method identifies royal jelly based on the protein sources in the feed, supplementing the original method of identifying based on the carbohydrate sources in the feed.

The present invention adopts the following technical solutions:

Taking royal jelly as the research object, an acetonitrile solution was used to extract daidzein in royal jelly (see Figure 1 for its structure), and then accurate qualitative and quantitative analysis was performed using liquid chromatography tandem mass spectrometry, and the feeding method of royal jelly was identified based on the analysis results. .

Preparation of standard stock solution (1000 µg/mL): Accurately weigh daidzein standard (equivalent to 10 mg of the target compound), dissolve it in chromatographically pure methanol, and dilute to 10 mL to obtain a standard stock solution with a concentration of 1000 µg/mL. The solution can be stored in a -20°C refrigerator away from light for 6 months. Preparation of standard intermediate solution (10 µg/mL): Accurately transfer 1 mL of the standard stock solution into a 100 mL volumetric flask, and dilute to 100 mL with chromatographically pure methanol to obtain a standard intermediate solution with a concentration of 10 µg/mL. The solution is - It can be stored in a refrigerator at 20°C away from light for 3 months. Preparation of standard working solution: Take an appropriate amount of 10 µg/mL standard intermediate solution, dilute it to constant volume with chromatographically pure methanol, and prepare the following series of standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg /L, 10 µg/L, prepare immediately before use.

Royal jelly sample pretreatment method: Weigh 2g of royal jelly into a 50 mL centrifuge tube, add 5 mL of zinc acetate buffer solution (ρ=219g/L), vortex for 1-3 min, add 20 mL of acetonitrile, vortex for 1-3 min, and then add 1 g NaCl and 4g Na2SO4, vortex for 1-3 minutes, centrifuge at 8000r/min for 5-10min at 4°C; add 8mL of supernatant to a 15 mL centrifuge tube containing 400 mg PSA, 400 mgC18EC and 200 mg MgSO4, vortex Spin for 1-3 minutes, centrifuge at 8000r/min for 5-10min at 4°C; put 2mL of the supernatant into a 5mL centrifuge tube, blow nitrogen to dryness, add 1mL of 50% (v/v) methanol solution to reconstitute, and vortex for 1 -3min, ultrasonic for 1-3min, pass through 0.2 µm filter membrane, and wait for use on the machine.

Liquid chromatography tandem mass spectrometry conditions: mobile phase is 0.1% formic acid-water (A), acetonitrile (B); elution conditions are shown in Table 1; Agilent Poroshell 120 EC-C18 column (2.1 mm×100 mm, 2.7 µm), The column temperature is 30°C; the flow rate is 0.2 mL/min; the injection volume is 5.0 µL; electrospray ion source (ESI); positive ion scanning mode; multiple reaction monitoring (MRM) mode; nebulizer temperature: 200°C, fog Nebulizer flow rate: 15 L/min, nebulizer pressure 30 psi, capillary voltage: 3500 V, sheath flow gas temperature: 350 ℃, sheath flow gas flow rate: 11 L/min. The parent ion of the standard material daidzein (4′,7-dihydroxyisoflavone) is 255.0, the quantitative ion is 136.9 (collision energy is 32V), the qualitative ion is 65.2 (collision energy is 57V), the residence time is 3ms, and the transmission The voltage is 380V.

Table 1 Liquid chromatography gradient elution table

The measurement method is as follows:

Qualitative determination: According to the content of the substance to be measured in the royal jelly sample solution, select a standard working solution with a similar peak area and a sample solution of equal volume for injection. Co-identification by chromatographic retention time and mass spectrometric selected ions. The relative deviation of the retention time of the substance to be measured and that of the standard substance in the sample is not greater than 1%, and the difference in the relative abundance of its selected ions is not greater than 10%.

Quantitative determination: Take an appropriate amount of royal jelly sample solution and standard working solution of corresponding concentration, perform single-point calibration or multi-point calibration, and quantify by the integrated value of the chromatographic peak area. The response values of the substance to be measured in the standard working solution and the sample solution should be within the linear range of the instrument detection. The standard working solution should be inserted during the injection process of the sample solution to facilitate accurate quantification.

Results: The correlation coefficient of the standard curve was greater than 0.991, the recovery rate of daidzein ranged from 87.95 to 117.99%, the relative standard deviation was less than 10.39%, the detection limit was 0.07µg/kg, and the quantitation limit was 0.23µg/kg.

Identification method: The daidzein content in non-fed (soybean meal) and fed (soybean meal) royal jelly samples is shown in Table 2. No daidzein was detected in the non-fed royal jelly samples, and the daidzein content in the fed royal jelly samples was 1.1535-11.757µg/kg. Mann-Whitney U test analysis showed that there was a very significant difference in the content of daidzein in the royal jelly between the two groups (P<0.0001), as shown in Figure 3. Therefore, the difference in daidzein content can be used to identify feeding royal jelly. If the content is greater than the method quantitation limit (0.23µg/kg), it means feeding (soybean meal) royal jelly.

Table 2 Content of daidzein in non-fed and fed royal jelly samples (µg/kg)

Note: <LOQ means less than the method limit of quantification

The present invention adopts the following technical solutions:

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

Example 1 Method for identifying artificially fed royal jelly based on daidzein content

Preparation of standard stock solution (1000 µg/mL): Accurately weigh daidzein standard (equivalent to 10 mg of the target compound), dissolve it in chromatographically pure methanol, and dilute to 10 mL to obtain a standard stock solution with a concentration of 1000 µg/mL. The solution can be stored in a -20°C refrigerator away from light for 6 months. Preparation of standard intermediate solution (10 µg/mL): Accurately transfer 1 mL of the standard stock solution into a 100 mL volumetric flask, and dilute to 100 mL with chromatographically pure methanol to obtain a standard intermediate solution with a concentration of 10 µg/mL. The solution is - It can be stored in a refrigerator at 20°C away from light for 3 months. Preparation of standard working solution: Take an appropriate amount of 10 µg/mL standard intermediate solution, dilute it to constant volume with chromatographically pure methanol, and prepare the following series of standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg /L, 10 µg/L, prepare immediately before use.

Royal jelly sample pretreatment method: Weigh 2g of royal jelly into a 50 mL centrifuge tube, add 5 mL of zinc acetate buffer solution (ρ=219g/L), vortex for 1 min, add 20 mL of acetonitrile, vortex for 1 min, then add 1 g of NaCl and 4 g of Na ₂ SO ₄ , vortex for 1 min, and centrifuge at 8000 r/min for 5 min at 4°C; add 8 mL of the supernatant into a 15 mL centrifuge tube containing 400 mg PSA, 400 mg C18EC and 200 mg MgSO ₄ , and vortex for 1 min at 4°C. Centrifuge at 8000 r/min for 5 minutes; put 2 mL of the supernatant into a 5 mL centrifuge tube, blow with nitrogen until dry, add 1 mL of 50% (v/v) methanol solution to reconstitute, vortex for 1 min, sonicate for 1 min, and filter with 0.2 μm Film, wait for machine.

Liquid chromatography tandem mass spectrometry conditions: mobile phase is 0.1% formic acid-water (A), acetonitrile (B); elution conditions are shown in Table 1; Agilent Poroshell 120 EC-C18 column (2.1 mm×100 mm, 2.7 μm), The column temperature is 30°C; the flow rate is 0.2 mL/min; the injection volume is 5.0 μL; electrospray ion source (ESI); positive ion scanning mode; multiple reaction monitoring (MRM) mode; nebulizer temperature: 200°C, fog Nebulizer flow rate: 15 L/min, nebulizer pressure 30 psi, capillary voltage: 3500 V, sheath flow gas temperature: 350 ℃, sheath flow gas flow rate: 11 L/min. The parent ion of the standard material daidzein (4′,7-dihydroxyisoflavone) is 255.0, the quantitative ion is 136.9 (collision energy is 32V), the qualitative ion is 65.2 (collision energy is 57V), the residence time is 3ms, and the transmission The voltage is 380V.

Determination method: Take an appropriate amount of royal jelly sample solution and standard working solution of corresponding concentration, perform single-point calibration or multi-point calibration, and quantify by the integrated value of the chromatographic peak area. The response values of the substance to be measured in the standard working solution and the sample solution should be within the linear range of the instrument detection. The standard working solution should be inserted during the injection process of the sample solution to facilitate accurate quantification.

Results: The correlation coefficient of the standard curve was 0.992, the recovery rate of daidzein ranged from 89.93 to 112.03%, the relative standard deviation was less than 10.29%, the detection limit was 0.07µg/kg, and the quantification limit was 0.23µg/kg.

Identification method: Among the 30 royal jelly samples collected in Zhejiang, 15 were fed (soybean meal) royal jelly with a daidzein content of 0.945-4.726µg/kg, while 15 non-fed samples used camellia pollen as the natural honey powder source. None were detected. Therefore, daidzein content can be used to identify feeding (soybean meal) royal jelly.

Example 2 Method for identifying artificially fed royal jelly based on daidzein content

Preparation of standard stock solution (1000 µg/mL): Accurately weigh daidzein standard (equivalent to 10 mg of the target compound), dissolve it in chromatographically pure methanol, and dilute to 10 mL to obtain a standard stock solution with a concentration of 1000 µg/mL. The solution can be stored in a -20°C refrigerator away from light for 6 months. Preparation of standard intermediate solution (10 µg/mL): Accurately transfer 1 mL of the standard stock solution into a 100 mL volumetric flask, and dilute to 100 mL with chromatographically pure methanol to obtain a standard intermediate solution with a concentration of 10 µg/mL. The solution is - It can be stored in a refrigerator at 20°C away from light for 3 months. Preparation of standard working solution: Take an appropriate amount of 10 µg/mL standard intermediate solution, dilute it to constant volume with chromatographically pure methanol, and prepare the following series of standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg /L, 10 µg/L, prepare immediately before use.

Royal jelly sample pretreatment method: Weigh 2g of royal jelly into a 50 mL centrifuge tube, add 5 mL of zinc acetate buffer solution (ρ=219g/L), vortex for 1 min, add 20 mL of acetonitrile, vortex for 1 min, then add 1 g of NaCl and 4 g of Na ₂ SO ₄ , vortex for 1 minute, centrifuge at 8000 r/min for 5 minutes at 4°C; add 8 mL of supernatant to a 15 mL centrifuge tube containing 400 mg PSA, 400 mg C18EC and 200 mg MgSO ₄ , vortex for 1 min, 4 Centrifuge at 8000r/min for 5min at ℃; put 2mL of supernatant into a 5mL centrifuge tube, blow with nitrogen until it is dry, add 1mL of 50% (v/v) methanol solution to reconstitute, vortex for 1min, sonicate for 1min, and pass through 0.2 µm Filter membrane and wait for machine.

Liquid chromatography tandem mass spectrometry conditions: mobile phase is 0.1% formic acid-water (A), acetonitrile (B); elution conditions are shown in Table 1; Agilent Poroshell 120 EC-C18 column (2.1 mm×100 mm, 2.7 μm), The column temperature is 30°C; the flow rate is 0.2 mL/min; the injection volume is 5.0 μL; electrospray ion source (ESI); positive ion scanning mode; multiple reaction monitoring (MRM) mode; nebulizer temperature: 200°C, fog Nebulizer flow rate: 15 L/min, nebulizer pressure 30 psi, capillary voltage: 3500 V, sheath flow gas temperature: 350 ℃, sheath flow gas flow rate: 11 L/min. The parent ion of the standard material daidzein (4′,7-dihydroxyisoflavone) is 255.0, the quantitative ion is 136.9 (collision energy is 32V), the qualitative ion is 65.2 (collision energy is 57V), the residence time is 3ms, and the transmission The voltage is 380V.

Determination method: Take an appropriate amount of royal jelly sample solution and standard working solution of corresponding concentration, perform single-point calibration or multi-point calibration, and quantify by the integrated value of the chromatographic peak area. The response values of the substance to be measured in the standard working solution and the sample solution should be within the linear range of the instrument detection. The standard working solution should be inserted during the injection process of the sample solution to facilitate accurate quantification.

Results: The correlation coefficient of the standard curve was 0.993, the recovery rate of daidzein ranged from 87.95-95.22%, the relative standard deviation was less than 3.53%, the detection limit was 0.07µg/kg, and the quantitation limit was 0.23µg/kg.

Identification method: Among the 30 royal jelly samples collected in Hubei, 15 were fed (soybean meal) royal jelly with a daidzein content of 1.136-2.904µg/kg, while 15 non-fed samples used rape pollen as a natural honey powder source. None were detected. Therefore, daidzein content can be used to identify feeding (soybean meal) royal jelly.

Example 3 Method for identifying artificially fed royal jelly based on daidzein content

Preparation of standard stock solution (1000 µg/mL): Accurately weigh daidzein standard (equivalent to 10 mg of the target compound), dissolve it in chromatographically pure methanol, and dilute to 10 mL to obtain a standard stock solution with a concentration of 1000 µg/mL. The solution can be stored in a -20°C refrigerator away from light for 6 months. Preparation of standard intermediate solution (10 µg/mL): Accurately transfer 1 mL of the standard stock solution into a 100 mL volumetric flask, and dilute to 100 mL with chromatographically pure methanol to obtain a standard intermediate solution with a concentration of 10 µg/mL. The solution is - It can be stored in a refrigerator at 20°C away from light for 3 months. Preparation of standard working solution: Take an appropriate amount of 10 µg/mL standard intermediate solution, dilute it to constant volume with chromatographically pure methanol, and prepare the following series of standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg /L, 10 µg/L, prepare immediately before use.

Royal jelly sample pretreatment method: Weigh 2g of royal jelly into a 50 mL centrifuge tube, add 5 mL of zinc acetate buffer solution (ρ=219g/L), vortex for 1 min, add 20 mL of acetonitrile, vortex for 1 min, then add 1 g of NaCl and 4 g of Na ₂ SO ₄ , vortex for 1 min, and centrifuge at 8000 r/min for 5 min at 4°C; add 8 mL of the supernatant into a 15 mL centrifuge tube containing 400 mg PSA, 400 mg C18EC and 200 mg MgSO ₄ , and vortex for 1 min at 4°C. Centrifuge at 8000 r/min for 5 minutes; put 2 mL of the supernatant into a 5 mL centrifuge tube, blow with nitrogen until dry, add 1 mL of 50% (v/v) methanol solution to reconstitute, vortex for 1 min, sonicate for 1 min, and filter with 0.2 μm Film, wait for machine.

Liquid chromatography tandem mass spectrometry conditions: mobile phase is 0.1% formic acid-water (A), acetonitrile (B); elution conditions are shown in Table 1; Agilent HC-C18 chromatographic column (250 mm×4.6 mm, 5µm), column temperature is 30℃; flow rate is 0.2 mL/min; injection volume is 5.0 μL; electrospray ion source (ESI); positive ion scanning mode; multiple reaction monitoring (MRM) mode; nebulizer temperature: 200℃, nebulizer flow rate : 15 L/min, nebulizer pressure 30 psi, capillary voltage: 3500 V, sheath gas temperature: 350 ℃, sheath gas flow rate: 11 L/min. The parent ion of the standard material daidzein (4′,7-dihydroxyisoflavone) is 255.0, the quantitative ion is 136.9 (collision energy is 32V), the qualitative ion is 65.2 (collision energy is 57V), the residence time is 3ms, and the transmission The voltage is 380V.

Determination method: Take an appropriate amount of royal jelly sample solution and standard working solution of corresponding concentration, perform single-point calibration or multi-point calibration, and quantify by the integrated value of the chromatographic peak area. The response values of the substance to be measured in the standard working solution and the sample solution should be within the linear range of the instrument detection. The standard working solution should be inserted during the injection process of the sample solution to facilitate accurate quantification.

Results: The correlation coefficient of the standard curve was 0.991, the recovery rate of daidzein ranged from 100.15-117.99%, the relative standard deviation was less than 9.00%, the detection limit was 0.07µg/kg, and the quantitation limit was 0.23µg/kg.

Identification method: Among the 20 royal jelly samples collected in Jiangsu, the daidzein content in 10 fed (soybean meal) royal jelly was 1.291-6.931µg/kg, while the 10 non-fed samples with camellia pollen as the natural honey powder source None were detected. Therefore, daidzein content can be used to identify feeding (soybean meal) royal jelly.

The characteristic ion mass chromatogram (MRM) of daidzein is shown in Figure 2A~Figure 2D.

Example 4 Method for identifying artificially fed royal jelly based on daidzein content

Preparation of standard stock solution (1000 µg/mL): Accurately weigh daidzein standard (equivalent to 10 mg of the target compound), dissolve it in chromatographically pure methanol, and dilute to 10 mL to obtain a standard stock solution with a concentration of 1000 µg/mL. The solution can be stored in a -20°C refrigerator away from light for 6 months. Preparation of standard intermediate solution (10 µg/mL): Accurately transfer 1 mL of the standard stock solution into a 100 mL volumetric flask, and dilute to 100 mL with chromatographically pure methanol to obtain a standard intermediate solution with a concentration of 10 µg/mL. The solution is - It can be stored in a refrigerator at 20°C away from light for 3 months. Preparation of standard working solution: Take an appropriate amount of 10 µg/mL standard intermediate solution, dilute it to constant volume with chromatographically pure methanol, and prepare the following series of standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg /L, 10 µg/L, prepare immediately before use.

Royal jelly sample pretreatment method: Weigh 2g of royal jelly into a 50 mL centrifuge tube, add 5 mL of zinc acetate buffer solution (ρ=219g/L), vortex for 1 min, add 20 mL of acetonitrile, vortex for 1 min, then add 1 g of NaCl and 4 g of Na ₂ SO ₄ , vortex for 1 min, and centrifuge at 8000 r/min for 5 min at 4°C; add 8 mL of the supernatant into a 15 mL centrifuge tube containing 400 mg PSA, 400 mg C18EC and 200 mg MgSO ₄ , and vortex for 1 min at 4°C. Centrifuge at 8000r/min for 5min; put 2mL of the supernatant into a 5mL centrifuge tube, blow with nitrogen until dry, add 1mL of 50% (v/v) methanol solution to reconstitute, vortex for 1min, sonicate for 1min, filter through 0.2µm filter , waiting for the machine.

Liquid chromatography tandem mass spectrometry conditions: mobile phase is 0.1% formic acid-water (A), acetonitrile (B); elution conditions are shown in Table 1; Agilent Poroshell 120 EC-C18 column (2.1 mm×100 mm, 2.7 μm), The column temperature is 30°C; the flow rate is 0.2 mL/min; the injection volume is 5.0 μL; electrospray ion source (ESI); positive ion scanning mode; multiple reaction monitoring (MRM) mode; nebulizer temperature: 200°C, fog Nebulizer flow rate: 15 L/min, nebulizer pressure 30 psi, capillary voltage: 3500 V, sheath flow gas temperature: 350 ℃, sheath flow gas flow rate: 11 L/min. The parent ion of the standard material daidzein (4′,7-dihydroxyisoflavone) is 255.0, the quantitative ion is 136.9 (collision energy is 32V), the qualitative ion is 65.2 (collision energy is 57V), the residence time is 3ms, and the transmission The voltage is 380V.

Determination method: Take an appropriate amount of royal jelly sample solution and standard working solution of corresponding concentration, perform single-point calibration or multi-point calibration, and quantify by the integrated value of the chromatographic peak area. The response values of the substance to be measured in the standard working solution and the sample solution should be within the linear range of the instrument detection. The standard working solution should be inserted during the injection process of the sample solution to facilitate accurate quantification.

Results: The correlation coefficient of the standard curve was 0.992, the recovery rate of daidzein ranged from 89.93 to 112.03%, the relative standard deviation was less than 10.29%, the detection limit was 0.07µg/kg, and the quantification limit was 0.23µg/kg.

Identification method: Take 10 samples from each of the royal jelly samples collected from Sichuan, Qinghai, Hubei and Zhejiang, for a total of 40 samples. Among them, 20 were from fed (soybean meal) royal jelly samples from Hubei and Zhejiang, and 20 were from non-fed royal jelly samples from Sichuan and Qinghai using rape pollen as a natural honey powder source. The daidzein content in fed (soybean meal) royal jelly was 0.581-11.757µg/kg, but was not detected in non-fed royal jelly. Therefore, daidzein content can be used to identify feeding (soybean meal) royal jelly.

Example 5 Royal jelly sample pretreatment, optimization of liquid chromatography and mass spectrometry conditions

1. Optimization of the extraction stage

In the process of dissolving royal jelly samples, experiments compared the effects of pure water and zinc acetate solution on dissolving the matrix. The results showed that when pure water is used as the solvent, more impurity interference peaks will appear, affecting the accurate quantification of the target compound. When zinc acetate solution is used as a solvent, the high concentration of acid ions it provides can take away the hydration layer on the protein surface, causing the protein micelles to lose water, and then condense and precipitate out, which is the salting out effect; and the zinc acetate solution has The acidic pH provided is close to the isoelectric point of most proteins in royal jelly, which also accelerates the precipitation of proteins and has the effect of purifying the matrix. Therefore, zinc acetate solution was finally selected to precipitate proteins in royal jelly.

The selection of extraction solvent should follow the three principles of not reacting with the solute, not miscible or reacting with the original solvent, and the solubility of the solute in the extraction solvent is greater than the solubility in the original solvent. In the present invention, the extraction effects of four extractants, acetonitrile, ethyl acetate, acetonitrile: dichloromethane = 1:1 (v/v) and acetonitrile: dichloromethane = 3:1 (v/v), are compared. The results showed that at the addition concentration of 10 μg/L, the recovery rates of daidzein extracted using four extractants were 74%, 49%, 62% and 52% respectively, so acetonitrile was finally selected as the extraction solvent.

2. Optimization of the purification stage

Royal jelly is rich in proteins, lipids and organic acids, which may interfere with the extraction of test substances. In this experiment, three commercial purification packages developed by Agilent Company (all purchased from Agilent Company in the United States) were selected to compare their purification effects through the recovery rate of daidzein and the stability of the recovery rate. The three types of purification packages are:

a) 5982-4950 (50 mg PSA (N-propylethylenediamine) + 150 mg C18EC + 900 mg Na ₂ SO ₄ ), remove matrix interference such as polar organic salts, sugars, lipids and proteins;

b) 5982-5158 (400 mg PSA (N-propylethylenediamine) + 400 mg C18EC + 1200 mg MgSO ₄ ), remove polar organic acids, certain sugars, and most lipids;

c) 5982-1010 (EMR), a proprietary lipid removal sorbent, innovative sorbent that enables lipid removal while retaining target analytes.

The results show that the recovery rates of daidzein in the three purification packages at an added concentration of 10 μg/L were 63%, 91% and 57% respectively, so 5982-5158 (400 mg PSA (N-propylethylenediamine)) was finally selected. +400 mg C18EC+1200 mgMgSO ₄ ) Purification Pack.

3. Optimization of chromatographic conditions

Agilent Poroshell 120 EC-C18 was selected as the liquid phase separation chromatography column in the experiment. Its particle size is 2.7 μm, consisting of a 1.7 μm diameter solid core and a 0.5 μm thick porous outer layer. This small particle size packing has high column efficiency similar to sub-2 μm columns, but the column back pressure is 40%-50% lower. This high efficiency, high resolution column can be used in any type of liquid chromatography. The porous layer and solid core limit diffusion distance and increase separation speed, while the narrow particle size distribution improves column efficiency and resolution. This column supports high pressure, and multiple columns can be connected in series to achieve the highest resolution and column efficiency.

The composition of the mobile phase not only affects the retention time, response value and peak shape of the target compound, but also affects the ionization efficiency of the target compound and the type of added ions. The present invention optimizes the composition of the mobile phase. In the positive mode, the retention and peak shape of the target compound when pure water-acetonitrile, 0.1% formic acid water-acetonitrile and 0.1% pure water-0.1% acetonitrile are used as the mobile phase are compared, because Formic acid can provide abundant H ⁺ , so after adding formic acid to the water phase, the response value of the compound is significantly improved, the peak shape is sharp, and the separation is improved, so 0.1% formic acid water-acetonitrile was finally selected as the mobile phase.

4. Optimization of mass spectrometry conditions

In order to obtain the best response value in MRM mode, this experiment optimized parameters such as residence time, gas temperature, dry gas flow rate, atomizer pressure, capillary voltage, sheath gas temperature, sheath gas flow rate and nozzle voltage. Among them, the capillary voltage was optimized between 2000V and 3500V. Since the molecular weight of the derivatized compound is relatively large, incomplete fragmentation of molecular ion fragments is likely to occur under low voltage, so 3500V was selected as the optimal capillary voltage.

5. Selection of detection marker—didzein

In the early stage, samples of fed (soybean meal) and non-fed (soybean meal) royal jelly were collected, and phytoestrogens were screened and identified. It was found that the characteristic substance daidzein derived from soybean meal was present in the fed (soybean meal) royal jelly. Yuan. Existing artificial bee feeds were also measured, and high concentrations of daidzein (105.78~2851.90 μg/kg) were detected in currently commercially available soybean meal powder and full-price bee food, as shown in Table 3. Such ingredients are detected in bees’ natural food such as pollen and honey. Therefore, daidzein was finally used as the detection marker.

Table 3 Content of daidzein in commercially available bee feed (µg/kg)

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (3)

1. A method for identifying artificially fed royal jelly based on daidzein content, which is characterized by using acetonitrile to extract daidzein in royal jelly samples, and then using liquid chromatography tandem mass spectrometry to conduct qualitative and quantitative analysis. According to different The difference in daidzein content in the royal jelly produced by bees under the feeding method can be used to identify whether the bees are fed soybean meal feed; The method includes the following steps: S1. Standard solution preparation: Use methanol to prepare the following series of daidzein standard working solutions: 0.1 µg/L, 0.5 µg/L, 1.0 µg/L, 5.0 µg/L, 10 µg/L; S2. Preparation of test solution: Take 2g royal jelly sample, add 5mL of 219g/L zinc acetate buffer solution, vortex for 1-3 minutes, add 20mL of acetonitrile, vortex for 1-3 minutes, then add 1g of NaCl and 4g of Na ₂ SO ₄ , Vortex for 1-3 minutes, place the mixed solution into a 50 mL centrifuge tube, and centrifuge at 8000 r/min for 5-10 minutes at 4°C; add 8 mL of the supernatant into a 15 mL centrifuge tube containing 400 mg PSA, 400 mg C18EC, and 200 mg MgSO ₄ , vortex for 1-3min, centrifuge at 8000r/min for 5-10min at 4°C; take 2mL of the supernatant in a 5mL centrifuge tube, blow nitrogen to dryness, add 1mL of 50v/v% methanol solution to reconstitute, vortex for 1- 3 minutes, ultrasonic for 1-3 minutes, pass through a 0.2 µm filter membrane to obtain the test solution; S3. Liquid chromatography tandem mass spectrometry detection: Inject a series of daidzein standard working solutions and test solution respectively, and draw a standard curve with the chromatographic peak area as the ordinate and the concentration of the series of daidzein standard working solutions as the abscissa. , use the standard curve to quantify the sample; The detection conditions of liquid chromatography tandem mass spectrometry are: Liquid chromatography conditions are as follows: mobile phase A is 0.1v/v% formic acid aqueous solution, mobile phase B is acetonitrile; elution conditions are shown in Table 1; Agilent Poroshell 120 EC-C18 chromatographic column, 2.1 mm×100 mm, 2.7 µm, column The temperature is 30°C; the flow rate is 0.2mL/min; the injection volume is 5.0 µL; Table 1 Liquid chromatography gradient elution table ; The mass spectrometry conditions are as follows: electrospray ion source; positive ion scanning mode; multiple reaction monitoring mode; nebulizer temperature 200°C, nebulizer flow rate 15 L/min, nebulizer pressure 30 psi, capillary voltage 3500 V, sheath flow gas temperature 350℃, sheath gas flow rate 11 L/min. 2. The method according to claim 1, characterized in that the parent ion of daidzein detected by mass spectrometry is 255.0, the quantitative ion is 136.9, and the qualitative ion is 65.2. 3. The method according to claim 1 or 2, characterized in that the recovery rate of daidzein is 87.95-117.99%, the detection limit is 0.07µg/kg, and the quantitation limit is 0.23µg/kg.