CN116930386B - Method for identifying artificial feeding royal jelly based on daidzein content - Google Patents
Method for identifying artificial feeding royal jelly based on daidzein content Download PDFInfo
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- ZQSIJRDFPHDXIC-UHFFFAOYSA-N daidzein Chemical compound C1=CC(O)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZQSIJRDFPHDXIC-UHFFFAOYSA-N 0.000 title claims abstract description 149
- 229940109850 royal jelly Drugs 0.000 title claims abstract description 104
- 235000007240 daidzein Nutrition 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 48
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 78
- 241000257303 Hymenoptera Species 0.000 claims abstract description 16
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 15
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 6
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 75
- 239000000243 solution Substances 0.000 claims description 46
- 150000002500 ions Chemical class 0.000 claims description 44
- 239000012224 working solution Substances 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 24
- 239000012488 sample solution Substances 0.000 claims description 23
- 238000001514 detection method Methods 0.000 claims description 21
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 4
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- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 4
- CFNHVUGPXZUTRR-UHFFFAOYSA-N n'-propylethane-1,2-diamine Chemical compound CCCNCCN CFNHVUGPXZUTRR-UHFFFAOYSA-N 0.000 description 4
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 3
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- KUPHXIFBKAORGY-UHFFFAOYSA-N 2-amino-3-iodo-4-methylbenzoic acid Chemical compound CC1=CC=C(C(O)=O)C(N)=C1I KUPHXIFBKAORGY-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- QHBZHVUGQROELI-UHFFFAOYSA-N Royal Jelly acid Natural products OCCCCCCCC=CC(O)=O QHBZHVUGQROELI-UHFFFAOYSA-N 0.000 description 2
- 240000000111 Saccharum officinarum Species 0.000 description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 description 2
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- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
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- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
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- 239000007832 Na2SO4 Substances 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
- 229940038481 bee pollen Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 235000013339 cereals Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
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- 210000003026 hypopharynx Anatomy 0.000 description 1
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- 230000007365 immunoregulation Effects 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Jellies, Jams, And Syrups (AREA)
Abstract
The invention provides a method for identifying manually fed royal jelly based on daidzein content, which adopts acetonitrile to extract daidzein in a royal jelly sample, uses a liquid chromatography tandem mass spectrometry to carry out qualitative and quantitative analysis on the daidzein, and identifies whether to feed bees with feed containing soybean meal according to the content difference of daidzein in the royal jelly produced by bees in different feeding modes. The invention can identify the royal jelly fed manually according to the daidzein content, and the method has the advantages of simplicity, rapidness, accuracy and stability, supplements the existing identification method and makes the identification result more convincing. The invention provides a new method for identifying and feeding the royal jelly, which has important significance for relevant departments to the quality supervision of the royal jelly products, and is convenient for consumers to select proper products according to own preference.
Description
Technical Field
The invention belongs to the technical field of food medicine detection, and particularly relates to a method for identifying artificial feeding royal jelly based on daidzein content.
Background
Royal jelly is also called royal jelly, is a pasty substance secreted by the hypopharynx gland and the palate gland in the 5 th to 14 th days after birth of young worker bees, is food for the life of worker bees larva and queen bees, and has a service life of about 35 days when the worker bees eat the royal jelly for a short time, and the service life of the queen bees eating the royal jelly for a whole life can reach 1-5 years. The water content of the royal jelly is about 60% -70%, the crude protein content is about 12% -15%, the total sugar content is about 10% -16%, the lipid content is about 3% -6%, and in addition, the royal jelly also contains vitamins, salt, mature amino acids and other substances. Royal jelly has various biological activities, and is a natural food suitable for human health care. A large number of researches show that the royal jelly has an immunoregulation effect, is helpful for delaying natural immune senescence, and the special fatty acid 10-hydroxy-2-decenoic acid (10-HDA) in the royal jelly has a potential effect on the treatment of hypoimmunity and can effectively enhance antigen-specific immune response. In addition, the royal jelly has strong antibacterial activity and anti-aging effect. Meanwhile, the royal jelly treatment can significantly improve glucose steady state disturbance and inflammatory reaction caused by high-fat diet, and has anti-diabetes and anti-obesity activity.
Bee honey and pollen are sucked by bees to manufacture royal jelly, and when external climatic conditions or honey powder source conditions are unfavorable, bee farmers usually supply nutrition to the bee colony by supplementing artificial feed, so that the yield of bee products is maintained, and the raising cost is reduced. Common artificial feeds include sugar feeds that provide a source of carbohydrates such as: sucrose, and protein feeds that provide a protein source such as: bean pulp, vegetable proteins, yeast extract, etc. The international standard of Royal jelly issued by International Standards Organization (ISO) divides Royal jelly produced by different feeding methods into two types: type 1 bees eat only natural foods (pollen, nectar, and honey); type 2 bees eat natural foods and other nutrients (proteins, carbohydrates, etc.) and indicate delta for both types of royal jelly 13 The C value (C13/C12 isotope ratio), sucrose, pyranosyl sucrose, maltose and maltotriose index are different and are subjected to classification requirements. The existing research reports show that the method for identifying the royal jelly produced by different feeding modes is mainly characterized by analyzing the sugar content and delta in the royal jelly 13 Differences in C values. Wytrychowski et al have found that royal jelly sugar content and delta based on sugarcane or corn starch hydrolysate feeding 13 The C values are all significantly different from non-fed royal jelly. It was also found that royal jelly delta produced when honey is fed with sucrose or corn hydrolysate, whether or not protein feed is fed 13 The C value is as high as-17 per mill, and the content of maltose, maltotriose, sucrose and glucopyranosyl sucrose is also increased significantly.
The prior research reports show that the method for identifying feeding royal jelly mainly comprises the steps of measuring various sugar contents and delta in the royal jelly 13 C difference, wytrychowski et al, showed that exogenous sugar fed royal jelly had sucrose, pyranosyl sucrose, maltose and maltotriose content and delta 13 The C value is higher than that of natural feeding royal jelly. Wherein the content of the cane sugar in the royal jelly obtained by feeding the sugarcane or beet can be up to 7.7%, the content of the pyranosyl sucrose can be up to 1.7%, and the content of the pyranosyl sucrose is 3 times higher than that of the royal jelly obtained by feeding the natural royal jelly; cereal and corn starch waterThe maltose content of the royal jelly obtained by feeding the solution is 1.4-5.5%, which is 2-5 times of that of the naturally fed royal jelly; the content of the maltotriose is 0.3-1.7 percent, which is 2-8 times of that of the natural feeding royal jelly; royal jelly delta obtained by feeding sucrose or corn hydrolysate 13 The C value is up to-17% 13 The C value is-26.45 to-23.73 percent. However, it was found that the compositional differences were mainly derived from feeding the sugar diet, whereas feeding the protein diet had no significant effect on the above assay results. In the production process of royal jelly, sufficient protein source supply (natural protein source: bee pollen, protein feed: bean pulp, etc.) is not available. The existing method is difficult to meet the identification of the royal jelly produced by feeding artificial protein feed.
The bean pulp is cheap and easy to obtain, and the main raw material of the artificial protein feed for bees in the market of China is the bean pulp. Therefore, the influence of small molecular specific components in the artificial protein feed, especially soybean meal, on the components of the royal jelly is utilized to establish and identify the royal jelly of the artificial feeding protein feed, and the supplement of the existing identification method can be realized.
Disclosure of Invention
The invention aims to provide a method for identifying artificial feeding royal jelly based on daidzein content.
In order to achieve the aim, the invention provides a method for identifying the artificial feeding of the royal jelly based on the daidzein content, which adopts acetonitrile to extract the daidzein in a royal jelly sample, then uses a liquid chromatography tandem mass spectrometry to carry out qualitative and quantitative analysis on the daidzein, and identifies whether the bees are fed with bean pulp feed according to the daidzein content difference in the royal jelly produced by the bees in different feeding modes.
Further, the method comprises the steps of:
s1, preparing a standard solution: the following series of daidzein standard working solutions were prepared with methanol: 0.1 g/L, 0.5 mug/L, 1.0 mug/L, 5.0 mug/L and 10 mug/L;
s2, preparing a sample solution: weighing 2g of Lac Regis Apis, placing in 50mL centrifuge tube, adding 5mL of zinc acetate buffer solution (ρ=219 g/L), swirling for 1-3min, adding 20mL of acetonitrile, swirling for 1-3min,1g NaCl and 4g Na were added 2 SO 4 Vortex for 1-3min, and centrifuge at 8000r/min for 5-10min at 4deg.C; 8mL of the supernatant was taken and charged with 400 mg of PSA (N-propylethylenediamine), 400 mg of C18EC and 200 mg of MgSO 4 In the 15 mL centrifuge tube of (2), vortex for 1-3min, and centrifuge for 5-10min at 8000r/min at 4 ℃; taking 2mL of supernatant, putting into a 5mL centrifuge tube, blowing nitrogen to dry, adding 1 mL of 50% (v/v) methanol solution for re-dissolving, swirling for 1-3min, and performing ultrasonic treatment for 1-3min to obtain a solution, and adding 0.2µm filter membrane, get the solution of sample;
s3, liquid chromatography tandem mass spectrometry detection: taking serial daidzein standard working solution and sample solution, respectively sampling, drawing a standard curve by taking chromatographic peak area as ordinate and serial daidzein standard working solution concentration as abscissa, and quantifying the sample by using the standard curve.
The detection conditions of the liquid chromatography tandem mass spectrometry are as follows:
the liquid chromatography conditions were as follows: mobile phase A is 0.1v/v% formic acid water solution, mobile phase B is acetonitrile; the elution conditions are shown in Table 1; agilent Poroshell 120-EC-C18 chromatographic column, 2.1 mm ×100 mm,2.7 μm, column temperature of 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L.
TABLE 1 liquid chromatography gradient elution table
The mass spectrometry conditions were as follows: electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode; atomizer temperature 200 ℃, atomizer flow rate 15L/min, atomizer pressure 30 psi, capillary voltage 3500V, sheath flow gas temperature 350 ℃, sheath flow gas flow rate 11L/min.
In the method, the mass spectrum of daidzein is 255.0, the quantitative ion is 136.9, and the qualitative ion is 65.2.
According to the method, the recovery rate of daidzein is 87.95-117.99%, the detection limit is 0.07 mu g/kg, and the quantitative limit is 0.23 mu g/kg.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention firstly utilizes acetonitrile solution to extract flavonoid compounds from a royal jelly sample, and utilizes a liquid chromatography tandem mass spectrometer to carry out qualitative and quantitative analysis on the flavonoid compounds. The liquid chromatography-tandem mass spectrometry combined technology adopted by the invention is that liquid chromatography is used as a separation system, mass spectrometry is used as a detection system, and a mass spectrum is obtained by separating and ionizing the extracted and purified sample in the liquid chromatography and mass spectrometry part and then passing through a detector. The liquid chromatography-mass spectrometry combines the advantages of high separation capacity of the chromatograph on complex samples, high selectivity of the mass spectrum, high sensitivity and capability of providing relative molecular weight and structural information.
The invention can identify the feeding royal jelly according to the daidzein content, and the method has the advantages of simplicity, rapidness, accuracy and stability, and supplements the existing identification method, so that the identification result is more convincing.
The invention optimizes the extracting reagent of the royal jelly, and the result shows that the highest extracting efficiency can be obtained only by selecting acetonitrile as an extracting solvent and using a C18 purifying bag to sufficiently remove impurities.
And (IV) diluting the royal jelly sample solution by using methanol and water before analyzing by using a liquid chromatography tandem mass spectrometer, otherwise, generating asymmetry of a chromatogram to cause inaccurate quantification.
And (V) adding 0.1% formic acid into the mobile phase of chromatographic detection to provide an acidic environment for the object to be detected and provide protons, so that the ionization efficiency is improved.
And (six) in the process of analyzing the royal jelly sample solution, only two sub-ions are contained, and the difference of the relative abundance is not more than 10%. The daidzein as the test substance is identified as being contained only when the relative deviation of the retention time is not more than 1%.
The seventh invention provides a new method for identifying and feeding royal jelly, which has important significance for relevant departments to the quality supervision of royal jelly products, and is convenient for consumers to select proper products according to own preference.
Drawings
FIG. 1 shows the structural formula of daidzein.
FIGS. 2A to 2D show characteristic ion mass chromatograms (MRMs) of daidzein in Lac Regis Apis measured by liquid chromatography tandem mass spectrometry in a preferred embodiment of the present invention. Wherein fig. 2A shows qualitative ion/quantitative ion relative abundance ratios; FIG. 2B shows a characteristic ion mass chromatogram (MRM) of daidzein standard solution (2 μg/L); FIG. 2C shows a quantitative ion (255→136.9) mass chromatogram; fig. 2D shows a qualitative ion (255→65.2) mass chromatogram.
FIG. 3 shows daidzein content of royal jelly in different feeding modes in a preferred embodiment of the present invention. * ***: p <0.0001 very significant level.
Detailed Description
The invention provides a method for identifying artificial feeding royal jelly based on daidzein content, which utilizes acetonitrile solution to extract soybean isoflavone compounds from a royal jelly sample, then carries out qualitative and quantitative analysis on the soybean isoflavone compounds, and identifies whether to feed bean powder feed according to the daidzein content difference in the royal jelly produced by different feeding modes. The method is used for identifying the royal jelly according to protein sources in the feed, and supplements the original method for identifying the royal jelly according to carbohydrate sources in the feed.
The invention adopts the following technical scheme:
taking Lac Regis Apis as research object, extracting daidzein (structure shown in figure 1) from Lac Regis Apis with acetonitrile solution, and accurately performing qualitative and quantitative analysis by liquid chromatography tandem mass spectrometry to identify feeding mode of Lac Regis Apis.
Preparation of a standard stock solution (1000 [ mu ] g/mL): accurately weighing daidzein standard substance (10 mg reduced to target compound), dissolving in chromatographic pure methanol, and fixing volume to 10mL to obtain standard stock solution with concentration of 1000 μg/mL, wherein the solution can be stored in a refrigerator at-20deg.C in dark place for 6 months. Preparation of a standard intermediate solution (10 [ mu ] g/mL): accurately transferring the standard stock solution 1 mL into a 100mL volumetric flask, and fixing the volume to 100mL by using chromatographic pure methanol to obtain a standard intermediate solution with the concentration of 10 mug/mL, wherein the solution can be stored for 3 months in a refrigerator at the temperature of minus 20 ℃ in a dark place. Preparing a standard working solution: taking a proper amount of 10 mug/mL standard intermediate liquid, sequentially diluting and fixing the volume by using chromatographic pure methanol, and preparing the following series of standard working liquids: 0.1 Mu g/L, 0.5 mu g/L, 1.0 mu g/L, 5.0 mu g/L and 10 mu g/L, and is prepared immediately before use.
The pretreatment method of the royal jelly sample comprises the following steps: weighing 2g of royal jelly, putting into a 50mL centrifuge tube, adding 5mL of zinc acetate buffer solution (rho=219 g/L), swirling for 1-3min, adding 20mL of acetonitrile, swirling for 1-3min, adding 1g of NaCl and 4g of Na2SO4, swirling for 1-3min, and centrifuging at 8000r/min for 5-10min at 4 ℃; 8mL of supernatant is taken and added into a 15 mL centrifuge tube filled with 400 mg of PSA, 400 mg of C18EC and 200 mg of MgSO4, vortexed for 1-3min and centrifuged at 8000r/min for 5-10min at 4 ℃; taking 2mL of supernatant, putting into a 5mL centrifuge tube, blowing nitrogen to dry, adding 1 mL of 50% (v/v) methanol solution for re-dissolving, swirling for 1-3min, and performing ultrasonic treatment for 1-3min to obtain a solution, and adding 0.2µm filter membranes, and waiting for loading.
Liquid chromatography tandem mass spectrometry conditions: mobile phase 0.1% formic acid-water (a), acetonitrile (B); the elution conditions are shown in Table 1; agilent Poroshell 120-EC-C18 column (2.1 mm ×100 mm,2.7 μm) with column temperature of 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L; electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode; atomizer temperature: 200. c, atomizer flow rate: 15 L/min, atomizer pressure 30 psi, capillary voltage: 3500V, sheath flow temperature: 350. sheath flow rate: 11 L/min. The standard daidzein (4', 7-dihydroxyisoflavone) parent ion 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 voltage is 380V.
TABLE 1 liquid chromatography gradient elution table
The measurement method is as follows:
qualitative determination: and selecting standard working solution with similar peak areas and the same-volume reference sample injection of the sample solution according to the content of the substance to be detected in the royal jelly sample solution. The ions were selected for co-characterization by chromatographic retention times with mass spectrometry. The relative deviation of the retention time of the substance to be detected and the standard substance in the sample is not more than 1%, and the difference of the relative abundance of the selected ions is not more than 10%.
Quantitative determination: and respectively taking a proper amount of royal jelly sample solution and standard working solution with corresponding concentration, performing single-point calibration or multi-point calibration, and quantifying by using a chromatographic peak area integral value. The response values of the standard working solution and the substances to be detected in the sample solution are in the linear range of instrument detection, and the standard working solution is inserted in the sample solution sampling process so as to accurately quantify.
Results: the correlation coefficient of the standard curve is larger than 0.991, the recovery rate of daidzein is in the range of 87.95-117.99%, the relative standard deviation is smaller than 10.39%, the detection limit is 0.07 mug/kg, and the quantitative limit is 0.23 mug/kg.
The identification method comprises the following steps: the daidzein content in the non-feeding (soybean meal) and feeding (soybean meal) royal jelly samples is shown in table 2, no daidzein is detected in the non-feeding royal jelly samples, and the daidzein content in the feeding royal jelly samples is 1.1535-11.757 mug/kg. The mann-whitney U test analysis showed that there was a very significant difference in daidzein content (P < 0.0001) between the two groups of royal jelly, as shown in fig. 3. So that the difference of daidzein content can be used for identifying the feeding of the royal jelly, and the feeding of the (soybean meal) royal jelly is the feeding of the royal jelly when the content is more than the quantitative limit (0.23 mu g/kg) of the method.
TABLE 2 non-feeding and feeding daidzein content (μg/kg) in Lac Regis Apis samples
Note that: < LOQ means less than the method quantitation limit
The invention adopts the following technical scheme:
the following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
Example 1 identification of Royal jelly fed artificially based on daidzein content
Preparation of a standard stock solution (1000 [ mu ] g/mL): accurately weighing daidzein standard substance (10 mg reduced to target compound), dissolving in chromatographic pure methanol, and fixing volume to 10mL to obtain standard stock solution with concentration of 1000 μg/mL, wherein the solution can be stored in a refrigerator at-20deg.C in dark place for 6 months. Preparation of a standard intermediate solution (10 [ mu ] g/mL): accurately transferring the standard stock solution 1 mL into a 100mL volumetric flask, and fixing the volume to 100mL by using chromatographic pure methanol to obtain a standard intermediate solution with the concentration of 10 mug/mL, wherein the solution can be stored for 3 months in a refrigerator at the temperature of minus 20 ℃ in a dark place. Preparing a standard working solution: taking a proper amount of 10 mug/mL standard intermediate liquid, sequentially diluting and fixing the volume by using chromatographic pure methanol, and preparing the following series of standard working liquids: 0.1 Mu g/L, 0.5 mu g/L, 1.0 mu g/L, 5.0 mu g/L and 10 mu g/L, and is prepared immediately before use.
The pretreatment method of the royal jelly sample comprises the following steps: weighing 2g of Lac Regis Apis in 50mL centrifuge tube, adding 5mL of zinc acetate buffer solution (ρ=219 g/L), swirling for 1min, adding 20mL of acetonitrile, swirling for 1min, and adding 1g of NaCl and 4g of Na 2 SO 4 Vortex for 1min, and centrifuge at 8000r/min for 5min at 4deg.C; 8mL of the supernatant was taken and charged with 400 mg of PSA, 400 mg of C18EC and 200 mg of MgSO 4 In a 15 mL centrifuge tube of (2), centrifuging at 8000r/min for 5min at 4 ℃ by vortexing for 1 min; taking 2mL of supernatant, putting into a 5mL centrifuge tube, blowing nitrogen to dry, adding 1 mL of 50% (v/v) methanol solution for re-dissolution, swirling for 1min, and performing ultrasonic treatment for 1min to obtain a solution of 0.2µm filter membranes, and waiting for loading.
Liquid chromatography tandem mass spectrometry conditions: mobile phase 0.1% formic acid-water (a), acetonitrile (B); the elution conditions are shown in Table 1; agilent Poroshell 120A EC-C18 chromatographic column (2.1 mm ×100 mm,2.7 μm) at 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L; electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode; atomizer temperature: 200. c, atomizer flow rate: 15 L/min, atomizer pressure 30 psi, capillary voltage: 3500V, sheath flow temperature: 350. sheath flow rate: 11 L/min. The standard daidzein (4', 7-dihydroxyisoflavone) parent ion 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 voltage is 380V.
The measuring method comprises the following steps: and respectively taking a proper amount of royal jelly sample solution and standard working solution with corresponding concentration, performing single-point calibration or multi-point calibration, and quantifying by using a chromatographic peak area integral value. The response values of the standard working solution and the substances to be detected in the sample solution are in the linear range of instrument detection, and the standard working solution is inserted in the sample solution sampling process so as to accurately quantify.
Results: the standard curve correlation coefficient is 0.992, the recovery rate range of daidzein is 89.93-112.03%, the relative standard deviation is less than 10.29%, the detection limit is 0.07 mug/kg, and the quantitative limit is 0.23 mug/kg.
The identification method comprises the following steps: among 30 royal jelly samples collected in Zhejiang, 15 fed (soybean meal) royal jelly contains 0.945-4.726 mug/kg of daidzein, and 15 non-fed samples taking tea pollen as a natural honey powder source are not detected. Therefore, the daidzein content can be used for identifying the feeding (soybean meal) royal jelly.
Example 2 identification of method of artificially fed Lac Regis Apis based on daidzein content
Preparation of a standard stock solution (1000 [ mu ] g/mL): accurately weighing daidzein standard substance (10 mg reduced to target compound), dissolving in chromatographic pure methanol, and fixing volume to 10mL to obtain standard stock solution with concentration of 1000 μg/mL, wherein the solution can be stored in a refrigerator at-20deg.C in dark place for 6 months. Preparation of a standard intermediate solution (10 [ mu ] g/mL): accurately transferring the standard stock solution 1 mL into a 100mL volumetric flask, and fixing the volume to 100mL by using chromatographic pure methanol to obtain a standard intermediate solution with the concentration of 10 mug/mL, wherein the solution can be stored for 3 months in a refrigerator at the temperature of minus 20 ℃ in a dark place. Preparing a standard working solution: taking a proper amount of 10 mug/mL standard intermediate liquid, sequentially diluting and fixing the volume by using chromatographic pure methanol, and preparing the following series of standard working liquids: 0.1 Mu g/L, 0.5 mu g/L, 1.0 mu g/L, 5.0 mu g/L and 10 mu g/L, and is prepared immediately before use.
The pretreatment method of the royal jelly sample comprises the following steps: weighing 2g of Lac Regis Apis in 50mL centrifuge tube, adding 5mL of zinc acetate buffer solution (ρ=219 g/L), swirling for 1min, adding 20mL of acetonitrile, swirling for 1min, and adding 1g of NaCl and 4g of Na 2 SO 4 Vortex for 1min, at 4deg.CCentrifuging at 8000r/min for 5min; 8mL of the supernatant was taken and charged with 400 mg of PSA, 400 mg of C18EC and 200 mg of MgSO 4 In a 15 mL centrifuge tube of (2), vortexing for 1min, centrifuging for 5min at 8000r/min at 4 ℃; taking 2mL of supernatant, putting into a 5mL centrifuge tube, blowing nitrogen to dry, adding 1 mL of 50% (v/v) methanol solution for re-dissolution, swirling for 1min, and performing ultrasonic treatment for 1min to obtain a solution of 0.2µm filter membranes, and waiting for loading.
Liquid chromatography tandem mass spectrometry conditions: mobile phase 0.1% formic acid-water (a), acetonitrile (B); the elution conditions are shown in Table 1; agilent Poroshell 120A EC-C18 chromatographic column (2.1 mm ×100 mm,2.7 μm) at 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L; electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode; atomizer temperature: 200. c, atomizer flow rate: 15 L/min, atomizer pressure 30 psi, capillary voltage: 3500V, sheath flow temperature: 350. sheath flow rate: 11 L/min. The standard daidzein (4', 7-dihydroxyisoflavone) parent ion 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 voltage is 380V.
The measuring method comprises the following steps: and respectively taking a proper amount of royal jelly sample solution and standard working solution with corresponding concentration, performing single-point calibration or multi-point calibration, and quantifying by using a chromatographic peak area integral value. The response values of the standard working solution and the substances to be detected in the sample solution are in the linear range of instrument detection, and the standard working solution is inserted in the sample solution sampling process so as to accurately quantify.
Results: the correlation coefficient of the standard curve is 0.993, the recovery rate range of daidzein is 87.95-95.22%, the relative standard deviation is less than 3.53%, the detection limit is 0.07 mug/kg, and the quantitative limit is 0.23 mug/kg.
The identification method comprises the following steps: of 30 royal jelly samples collected in Hubei, 15 fed (soybean meal) royal jelly contains 1.136-2.904 mug/kg of daidzein, and 15 non-fed samples taking rape pollen as a natural honey powder source are not detected. Therefore, the daidzein content can be used for identifying the feeding (soybean meal) royal jelly.
Example 3 identification of method for artificially feeding Lac Regis Apis based on daidzein content
Preparation of a standard stock solution (1000 [ mu ] g/mL): accurately weighing daidzein standard substance (10 mg reduced to target compound), dissolving in chromatographic pure methanol, and fixing volume to 10mL to obtain standard stock solution with concentration of 1000 μg/mL, wherein the solution can be stored in a refrigerator at-20deg.C in dark place for 6 months. Preparation of a standard intermediate solution (10 [ mu ] g/mL): accurately transferring the standard stock solution 1 mL into a 100mL volumetric flask, and fixing the volume to 100mL by using chromatographic pure methanol to obtain a standard intermediate solution with the concentration of 10 mug/mL, wherein the solution can be stored for 3 months in a refrigerator at the temperature of minus 20 ℃ in a dark place. Preparing a standard working solution: taking a proper amount of 10 mug/mL standard intermediate liquid, sequentially diluting and fixing the volume by using chromatographic pure methanol, and preparing the following series of standard working liquids: 0.1 Mu g/L, 0.5 mu g/L, 1.0 mu g/L, 5.0 mu g/L and 10 mu g/L, and is prepared immediately before use.
The pretreatment method of the royal jelly sample comprises the following steps: weighing 2g of Lac Regis Apis in 50mL centrifuge tube, adding 5mL of zinc acetate buffer solution (ρ=219 g/L), swirling for 1min, adding 20mL of acetonitrile, swirling for 1min, and adding 1g of NaCl and 4g of Na 2 SO 4 Vortex for 1min, and centrifuge at 8000r/min for 5min at 4deg.C; 8mL of the supernatant was taken and charged with 400 mg of PSA, 400 mg of C18EC and 200 mg of MgSO 4 In a 15 mL centrifuge tube of (2), centrifuging at 8000r/min for 5min at 4 ℃ by vortexing for 1 min; taking 2mL of supernatant, putting into a 5mL centrifuge tube, blowing nitrogen to dry, adding 1 mL of 50% (v/v) methanol solution for re-dissolution, swirling for 1min, and performing ultrasonic treatment for 1min to obtain a solution of 0.2µm filter membranes, and waiting for loading.
Liquid chromatography tandem mass spectrometry conditions: mobile phase 0.1% formic acid-water (a), acetonitrile (B); the elution conditions are shown in Table 1; agilent HC-C18 chromatographic column (250 mm ×4.6 mm,5 μm) with column temperature of 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L; electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode; atomizer temperature: 200. c, atomizer flow rate: 15 L/min, atomizer pressure 30 psi, capillary voltage: 3500V, sheath flow temperature: 350. sheath flow rate: 11 L/min. The standard daidzein (4', 7-dihydroxyisoflavone) parent ion 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 voltage is 380V.
The measuring method comprises the following steps: and respectively taking a proper amount of royal jelly sample solution and standard working solution with corresponding concentration, performing single-point calibration or multi-point calibration, and quantifying by using a chromatographic peak area integral value. The response values of the standard working solution and the substances to be detected in the sample solution are in the linear range of instrument detection, and the standard working solution is inserted in the sample solution sampling process so as to accurately quantify.
Results: the standard curve correlation coefficient is 0.991, the recovery rate range of daidzein is 100.15-117.99%, the relative standard deviation is less than 9.00%, the detection limit is 0.07 mug/kg, and the quantitative limit is 0.23 mug/kg.
The identification method comprises the following steps: among 20 royal jelly samples collected from Jiangsu, 10 fed (soybean meal) royal jelly contains 1.291-6.931 mug/kg of daidzein, and 10 non-fed samples taking tea pollen as a natural honey powder source are not detected. Therefore, the daidzein content can be used for identifying the feeding (soybean meal) royal jelly.
The characteristic ion mass chromatogram (MRM) of daidzein is shown in figures 2A-2D.
Example 4 identification of method for artificially feeding Lac Regis Apis based on daidzein content
Preparation of a standard stock solution (1000 [ mu ] g/mL): accurately weighing daidzein standard substance (10 mg reduced to target compound), dissolving in chromatographic pure methanol, and fixing volume to 10mL to obtain standard stock solution with concentration of 1000 μg/mL, wherein the solution can be stored in a refrigerator at-20deg.C in dark place for 6 months. Preparation of a standard intermediate solution (10 [ mu ] g/mL): accurately transferring the standard stock solution 1 mL into a 100mL volumetric flask, and fixing the volume to 100mL by using chromatographic pure methanol to obtain a standard intermediate solution with the concentration of 10 mug/mL, wherein the solution can be stored for 3 months in a refrigerator at the temperature of minus 20 ℃ in a dark place. Preparing a standard working solution: taking a proper amount of 10 mug/mL standard intermediate liquid, sequentially diluting and fixing the volume by using chromatographic pure methanol, and preparing the following series of standard working liquids: 0.1 Mu g/L, 0.5 mu g/L, 1.0 mu g/L, 5.0 mu g/L and 10 mu g/L, and is prepared immediately before use.
The pretreatment method of the royal jelly sample comprises the following steps: weighing 2g of Lac Regis Apis, adding 5mL of zinc acetate buffer solution (ρ=219 g/L) into 50mL centrifuge tube, swirling for 1min, adding 20mL acetonitrile, vortex for 1min, add 1g NaCl and 4g Na 2 SO 4 Vortex for 1min, and centrifuge at 8000r/min for 5min at 4deg.C; 8mL of the supernatant was taken and charged with 400 mg of PSA, 400 mg of C18EC and 200 mg of MgSO 4 In a 15 mL centrifuge tube of (2), centrifuging at 8000r/min for 5min at 4 ℃ by vortexing for 1 min; taking 2mL of supernatant in a 5mL centrifuge tube, blowing nitrogen to be dry, adding 1 mL of 50% (v/v) methanol solution for re-dissolution, swirling for 1min, performing ultrasonic treatment for 1min, and passing through a 0.2 mu m filter membrane to be put on the machine.
Liquid chromatography tandem mass spectrometry conditions: mobile phase 0.1% formic acid-water (a), acetonitrile (B); the elution conditions are shown in Table 1; agilent Poroshell 120A EC-C18 chromatographic column (2.1 mm ×100 mm,2.7 μm) at 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L; electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode; atomizer temperature: 200. c, atomizer flow rate: 15 L/min, atomizer pressure 30 psi, capillary voltage: 3500V, sheath flow temperature: 350. sheath flow rate: 11 L/min. The standard daidzein (4', 7-dihydroxyisoflavone) parent ion 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 voltage is 380V.
The measuring method comprises the following steps: and respectively taking a proper amount of royal jelly sample solution and standard working solution with corresponding concentration, performing single-point calibration or multi-point calibration, and quantifying by using a chromatographic peak area integral value. The response values of the standard working solution and the substances to be detected in the sample solution are in the linear range of instrument detection, and the standard working solution is inserted in the sample solution sampling process so as to accurately quantify.
Results: the standard curve correlation coefficient is 0.992, the recovery rate range of daidzein is 89.93-112.03%, the relative standard deviation is less than 10.29%, the detection limit is 0.07 mug/kg, and the quantitative limit is 0.23 mug/kg.
The identification method comprises the following steps: 10 samples of royal jelly were taken from Sichuan, qinghai, hubei and Zhejiang respectively, and 40 samples were taken in total. Of these, 20 were from Hubei and Zhejiang feeding (soybean meal) Lac Regis Apis samples, and 20 were from Sichuan and Qinghai non-feeding Lac Regis Apis samples with rape pollen as natural honey powder source. The daidzein content in the feeding (soybean meal) royal jelly is 0.581-11.757 mug/kg, but is not detected in the feeding royal jelly. Therefore, the daidzein content can be used for identifying the feeding (soybean meal) royal jelly.
EXAMPLE 5 pretreatment of Royal jelly sample, liquid chromatography and optimization of Mass Spectrometry conditions
1. Optimization of extraction stage
In the process of dissolving the royal jelly sample, the effect of dissolving the matrix by pure water and zinc acetate solution is compared through experiments, and the result shows that more impurity interference peaks can appear when pure water is used as a solvent, and the accurate quantification of the target compound is affected. When the zinc acetate solution is used as a solvent, the high-concentration acid radical ions provided by the zinc acetate solution can deprive a hydration layer on the surface of protein, so that protein colloidal particles lose water, and then are coagulated and precipitated, namely, a salting-out effect is achieved; and the acidic pH provided by the zinc acetate solution is close to the isoelectric point of most proteins in the royal jelly, so that the precipitation of the proteins is accelerated, and the effect of purifying the matrix is achieved, so that the zinc acetate solution is finally selected for precipitating the proteins in the royal jelly.
The extraction solvent is selected according to three principles that the extraction solvent does not react with the solute, is not mutually soluble or reacts with the original solvent, and the solubility of the solute in the extraction solvent is greater than the solubility in the original solvent. Acetonitrile, ethyl acetate, acetonitrile were compared in the present invention: dichloromethane = 1:1 (v/v) and acetonitrile: dichloromethane = 3:1 (v/v) extraction effect of four extractants. The results showed that at an added concentration of 10. Mu.g/L, the recovery rates of daidzein extracted with the four extractants were 74%, 49%, 62% and 52%, respectively, and therefore acetonitrile was finally selected for use as the extraction solvent.
2. Optimization of the purification phase
The Lac Regis Apis contains abundant proteins, lipids and organic acids, and has interference effect on the extraction of the analyte. In this experiment, 3 commercial purification packages (all purchased from Agilent corporation in usa) developed by Agilent corporation were selected, and the purification effect was compared by the recovery rate of daidzein and the stability of recovery rate. The three purification packages are respectively:
a) 5982-4950 (50 mg PSA (N-propyl)Ethylenediamine) +150 mg C18EC+900 mg Na 2 SO 4 ) Removing matrix interference such as polar organic salts, saccharides, lipids, proteins and the like;
b) 5982-5158 (400 mg PSA (N-propylethylenediamine) +400 mg C18EC+1200 mg MgSO) 4 ) Removing polar organic acids, certain saccharides, and most of lipids;
c) 5982-1010 (EMR), proprietary lipid removal adsorbents, innovative adsorbents capable of achieving lipid removal while retaining target analytes.
The results showed that the recovery rates of daidzein for the three purification packages at 10. Mu.g/L added concentration were 63%, 91% and 57%, respectively, so that 5982-5158 (400 mg PSA (N-propylethylenediamine) +400 mg C18EC+1200 mg MgSO) was finally selected 4 ) And (5) purifying the bag.
3. Optimization of chromatographic conditions
Agilent Porosiwell 120 EC-C18 was chosen for the liquid separation column. The particle size was 2.7. Mu.m, consisting of a solid core of 1.7 μm diameter and a porous outer layer of 0.5. Mu.m. Such small particle size packing has a high column efficiency similar to a sub-2 μm column, but a column reverse pressure of 40% -50% lower. The high-column efficiency and high-separation chromatographic column can be used for any type of liquid chromatography. The porous layer and the solid core limit the diffusion distance, increase the separation speed, and the narrow particle size distribution increases the column efficiency and the separation degree. The column supports high pressure, and can realize highest separation degree and column efficiency by adopting multi-column series connection.
The composition of the mobile phase affects not only the retention time, response and peak shape of the target compound, but also the ionization efficiency of the target compound and the type of ions added. The present invention optimizes the composition of the mobile phase and compares the retention and peak shape of the target compound when pure water-acetonitrile, 0.1% formic acid-acetonitrile and 0.1% pure water-0.1% acetonitrile are used as mobile phases in the positive mode because formic acid can provide abundant H + Therefore, after formic acid in the aqueous phase, the response value of the compound is obviously improved, the peak shape is sharp, and the separation degree is improved, so that 0.1% of formic acid water-acetonitrile is finally selected as a mobile phase.
4. Optimization of mass spectrometry conditions
In order to obtain the best response value in the MRM mode, the experiment optimizes parameters such as residence time, gas temperature, dry gas flow rate, atomizer pressure, capillary voltage, sheath gas temperature, sheath gas flow rate, nozzle voltage and the like. The capillary voltage is optimized between 2000V and 3500V, and the molecular weight of the derivative compound is large, so that incomplete fragmentation of molecular ion fragments is easily caused under low voltage, and 3500V is selected as the optimal capillary voltage.
5. Selection of the detection marker daidzein
In the early stage, samples of feeding (soybean meal) and non-feeding (soybean meal) royal jelly are collected, phytoestrogens in the samples are screened and identified, and the characteristic substance daidzein derived from the soybean meal exists in the feeding (soybean meal) royal jelly. The conventional artificial feed for bees was also measured, and daidzein (105.78 to 2851.90. Mu.g/kg) was detected at a high concentration in both the soybean meal powder and the whole bee bread, which are currently commercially available, as shown in Table 3. The components are detected in bee natural foods such as pollen, honey and the like. Therefore, daidzein is ultimately used as a detection marker.
TABLE 3 daidzein content in commercial bee feeds (μg/kg)
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (3)
1. A method for identifying the artificial feeding of royal jelly based on daidzein content is characterized in that acetonitrile is adopted to extract daidzein in a royal jelly sample, then qualitative and quantitative analysis is carried out on the daidzein by utilizing a liquid chromatography tandem mass spectrometry, and whether the bees are fed with bean flour feed is identified according to the difference of the daidzein content in the royal jelly produced by the bees in different feeding modes;
the method comprises the following steps:
s1, preparing a standard solution: the following series of daidzein standard working solutions were prepared with methanol: 0.1 g/L, 0.5 mug/L, 1.0 mug/L, 5.0 mug/L and 10 mug/L;
s2, preparing a sample solution: taking 2g of royal jelly sample, adding 219g/L zinc acetate buffer solution 5mL, swirling for 1-3min, adding 20mL acetonitrile, swirling for 1-3min, and adding 1g NaCl and 4g Na 2 SO 4 Swirling for 1-3min, placing the mixed solution into a 50mL centrifuge tube, and centrifuging for 5-10min at 8000r/min at 4 ℃; 8mL of supernatant was taken and added with 400 mg of PSA, 400 mg of C18EC and 200 mg of MgSO 4 In the 15 mL centrifuge tube of (2), vortex for 1-3min, and centrifuge for 5-10min at 8000r/min at 4 ℃; taking 2mL of supernatant, putting into a 5mL centrifuge tube, blowing nitrogen to dry, adding 1 mL of 50v/v% methanol solution for re-dissolving, swirling for 1-3min, and performing ultrasonic treatment for 1-3min to obtain a solution, and adding 0.2µm filter membrane, get the solution of sample;
s3, liquid chromatography tandem mass spectrometry detection: taking serial daidzein standard working solution and sample solution, respectively sampling, drawing a standard curve by taking chromatographic peak area as ordinate and serial daidzein standard working solution concentration as abscissa, and quantifying the sample by using the standard curve;
the detection conditions of the liquid chromatography tandem mass spectrometry are as follows:
the liquid chromatography conditions were as follows: mobile phase A is 0.1v/v% formic acid water solution, mobile phase B is acetonitrile; the elution conditions are shown in Table 1; agilent Poroshell 120-EC-C18 chromatographic column, 2.1 mm ×100 mm,2.7 μm, column temperature of 30deg.C; the flow rate is 0.2 mL/min; the sample injection amount is 5.0 mu L;
TABLE 1 liquid chromatography gradient elution table
；
The mass spectrometry conditions were as follows: an electrospray ion source; a positive ion scanning mode; a multiple reaction monitoring mode; atomizer temperature 200 ℃, atomizer flow rate 15L/min, atomizer pressure 30 psi, capillary voltage 3500V, sheath flow gas temperature 350 ℃, sheath flow gas flow rate 11L/min.
2. The method of claim 1, wherein the daidzein has a parent ion of 255.0, a quantitative ion of 136.9, and a qualitative ion of 65.2 as detected by mass spectrometry.
3. The method according to claim 1 or 2, characterized in that the recovery of daidzein is 87.95-117.99%, the detection limit is 0.07 μg/kg and the quantification limit is 0.23 μg/kg.
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Non-Patent Citations (8)
| Title |
|---|
| Changes in the chemical composition of royal jelly produced through artificial bee-feeding in response to seasonal variations during non-migratory beekeeping;Kim, Eunhee 等;Journal of Food Composition & Analysis;第115卷;第104982页 * |
| Determination of Trans-10-Hydroxy-2-Decenoic Acid in Royal Jelly by High Performance Liquid Chromatography after Different Bee Feeding;Ralitsa Balkanska;International Journal of Current Microbiology and Applied Sciences;第04卷(第04期);第3738-3743页 * |
| Honeybee nutrition and pollen substitutes: A review;Bilal Ahamad Paray 等;Saudi Journal of Biological Sciences;第28卷(第01期);第1167-1176页 * |
| Optimization and application of parallel solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry for the determination of 11 aminoglycoside residues in honey and royal jelly;Xinran Wang 等;Journal of Chromatography A;第1542卷;第28-36页 * |
| Physicochemical characterisation of French royal jelly: Comparison with commercial royal jellies and royal jellies produced through artificial bee-feeding;Wytrychowski, M. 等;Journal of Food Composition and Analysis;第29卷(第03期);第126-133页 * |
| Soy extract as protein replacement to feed Melipona flavolineata Friese (Hymenoptera, Apidae, Meliponini;Joyce Teixeira 等;Journal of Apicultural Research;第59卷(第01期);第104-114页 * |
| 超高效液相色谱－串联质谱法测定蜂蜜中 24种酚酸和黄酮类化合物;张敏 等;分析测试学报;第41卷(第11期);第1636-1643页 * |
| 饲料蛋白源对意大利蜜蜂繁蜂效果及蜂王浆品质的影响;李肖;中国优秀硕士学位论文全文数据库 (农业科技辑)(第01期);第D051-17页 * |
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