CN114878710A - Method for detecting content of hydroxymethyl furfural in royal jelly and dry powder thereof - Google Patents
Method for detecting content of hydroxymethyl furfural in royal jelly and dry powder thereof Download PDFInfo
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- CN114878710A CN114878710A CN202210477234.XA CN202210477234A CN114878710A CN 114878710 A CN114878710 A CN 114878710A CN 202210477234 A CN202210477234 A CN 202210477234A CN 114878710 A CN114878710 A CN 114878710A
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- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 title claims abstract description 102
- 229940109850 royal jelly Drugs 0.000 title claims abstract description 100
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000000843 powder Substances 0.000 title claims abstract description 33
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 114
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000001514 detection method Methods 0.000 claims abstract description 27
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 24
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 238000010812 external standard method Methods 0.000 claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 81
- 239000000523 sample Substances 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 45
- 239000012496 blank sample Substances 0.000 claims description 41
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 38
- 239000012488 sample solution Substances 0.000 claims description 25
- 239000012086 standard solution Substances 0.000 claims description 25
- 239000012224 working solution Substances 0.000 claims description 20
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 19
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 9
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- 238000012856 packing Methods 0.000 claims description 5
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- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 3
- 238000011084 recovery Methods 0.000 abstract description 7
- 238000007865 diluting Methods 0.000 abstract description 2
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- 238000000605 extraction Methods 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000012088 reference solution Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000257303 Hymenoptera Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
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- 239000011550 stock solution Substances 0.000 description 3
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000000276 potassium ferrocyanide Substances 0.000 description 2
- 230000006920 protein precipitation Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004611 spectroscopical analysis Methods 0.000 description 2
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 2
- 239000004246 zinc acetate Substances 0.000 description 2
- QVYAWBLDJPTXHS-UHFFFAOYSA-N 5-Hydroxymethyl-2-furfural Natural products OC1=CC=C(C=O)O1 QVYAWBLDJPTXHS-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 241000256844 Apis mellifera Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229920002302 Nylon 6,6 Polymers 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
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- 210000004080 milk Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
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- 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
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- 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
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- 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
- G01N30/14—Preparation by elimination of some components
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- 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
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N30/52—Physical parameters
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Abstract
The invention discloses a method for measuring the content of hydroxymethyl furfural in royal jelly and dry powder thereof, which comprises the steps of dissolving and diluting a sample by water and 3% ammonia water, extracting the hydroxymethyl furfural by ethyl acetate, analyzing by a high performance liquid chromatograph, and quantifying by an external standard method. The method has detection limit of 3-6 μ g/kg, quantitative limit of 10-20 μ g/kg, recovery rate of 82-106% (n ═ 3), and precision of 4.6-8.0% (n ═ 3), and can meet the requirement for determining freshness of royal jelly and royal jelly dry powder.
Description
Technical Field
The invention belongs to the field of food quality control, and relates to a detection method of hydroxymethylfurfural as an index of freshness in royal jelly and dry powder thereof.
Background
Royal jelly (royal jelly), which is the secretion of pharyngeal gland of young worker bees that breed larvae in honeybee hives, is a food for larvae that will become queen bees and also a food for the queen bees to live for their lifetime. Proved by years of scientific research and medical clinical practice at home and abroad, the royal jelly has peculiar efficacy on human medical treatment, health care and the like.
The royal jelly contains 62-67% of water, 14-16% of protein, 10-15% of saccharide, 2-9% of lipid, 0.6-2% of mineral substances, and trace vitamins. In the production practice, if the royal jelly is not stored properly, the royal jelly can generate browning, become sticky due to aggregation and solidification of protein, become sour and smelly due to microbial contamination and generate bubbles, thereby seriously affecting the quality of the royal jelly.
The 2008 patent publication No. CN101363828B proposes that the royal jelly belongs to a fresh grade when the content of 5-hydroxymethyl-2-furfural in the royal jelly is less than or equal to 90 mug/kg. The freshness index can stimulate the production of high quality royal jelly. However, the method of the patent is operated, because the sample is diluted by 20 times, the detection limit of the method is reduced, and the detection limit of the method is 0.3mg/kg through measurement, so that the requirement of the quality control of the freshness of the royal jelly cannot be met.
Hydroxymethylfurfural is an important intermediate product of the maillard reaction, and is formed by the dehydration of hexoses under acidic conditions, or the reaction between amino acids and carbonyl compounds. Journal of Spectroscopy and Spectroscopy, 2009,29(12):3236-3240, indicates that the content of hydroxymethylfurfural in royal jelly is below 150 μ g/kg, and royal jelly can be considered to be fresh.
In the existing literature detection method, the detection limit of hydroxymethylfurfural in royal jelly is 300 mug/kg, and the quantitative limit is 1mg/kg, however, in our research, the royal jelly stored at 4 ℃ in 2019 has the hydroxymethylfurfural content of only 91 mug/kg, so that the method for measuring the hydroxymethylfurfural content in royal jelly needs to be further researched.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof. The method has the characteristics of simple, rapid, accurate and sensitive operation, common and easily-obtained instruments, convenience for popularization, high practicability and capability of providing a basis for determining the freshness index of the royal jelly, wherein the detection limit of the method reaches 6 mug/kg, and the quantification limit of the method reaches 20 mug/kg.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for detecting hydroxymethylfurfural content in royal jelly and dry powder thereof adopts high performance liquid chromatography and external standard method for quantification, and comprises the following steps:
1) determining chromatographic conditions:
chromatographic column packing agent: octadecyl bonded silica gel;
mobile phase: the mobile phase A is methanol-0.4% phosphoric acid solution, and the volume ratio is 0-10: 90-100; (ii) a Mobile phase B, methanol; detection wavelength: 285 nm; column temperature: 35 ℃;
2) preparation of a test solution:
weighing a royal jelly sample or a royal jelly dry powder sample, dissolving in an ammonia water solution, extracting with ethyl acetate, evaporating an ethyl acetate layer to dryness, adding a methanol solution, and filtering with a filter membrane to obtain a sample solution containing 1.00g of sample per 1 ml;
3) preparation of blank sample standard solution:
weighing 5 parts of blank sample, adding hydroxymethylfurfural standard working solution to obtain blank sample standard solution with the concentration of 20 mug/L, 50 mug/L, 100 mug/L, 200 mug/L and 500 mug/L;
4) and (3) determination:
respectively and precisely absorbing 20 mu L of blank sample standard solution and sample solution, injecting the blank sample standard solution and the sample solution into a liquid chromatograph, drawing a standard working curve by taking the concentration of the blank sample standard solution as a horizontal coordinate and the peak area of the hydroxymethylfurfural as a vertical coordinate, and quantifying by using an external standard method.
Further, the mobile phase A is methanol-0.4% phosphoric acid solution, and the volume ratio is 6: 94.
Further, the alkaline solution is selected from ammonia, an aqueous carbonate solution, an aqueous sodium hydroxide solution, and an aqueous calcium hydroxide solution so that the pH of the sample solution is 7 to 8.
Furthermore, the detection limit of the detection method for the content of the hydroxymethylfurfural in the royal jelly and the dry powder thereof is 3-6 mug/kg, and the quantitative limit is 10-20 mug/kg.
Specifically, the method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof adopts high performance liquid chromatography and an external standard method for quantification, and comprises the following steps:
1) determining chromatographic conditions:
chromatographic column packing agent: octadecyl bonded silica gel;
mobile phase: mobile phase A, methanol-0.4% phosphoric acid solution with the volume ratio of 6: 94; mobile phase B, methanol; detection wavelength: 285 nm; column temperature: 35 ℃;
2) preparing a test solution:
weighing 1.00g of royal jelly sample or 0.50g of royal jelly dry powder sample, accurately obtaining the sample with the sample weight of +/-0.001 g, adding 1.5mL of water into a 15mL plastic centrifuge tube, adding 1.5mL of 3% ammonia water solution with volume concentration into the royal jelly sample, adding 2.0mL of 3% ammonia water solution with volume concentration into the royal jelly dry powder sample, fully dissolving the mixture until royal jelly particles can not be seen by naked eyes, adding 8mL of ethyl acetate, manually oscillating and extracting for 1min, and carrying out centrifugal separation at the rotating speed of 4500 r/min for 5 min; sucking all ethyl acetate layers into a 50mL heart-shaped bottle, rotating in a water bath at 40 ℃ to evaporate to dryness, adding 1mL of methanol solution with the concentration of 6% into the heart-shaped bottle, dissolving residues by ultrasonic waves, and filtering the sample solution with a 0.22-micron filter membrane to obtain a sample solution containing 1.00g of sample per 1 mL;
3) preparation of blank sample standard solution:
weighing 5 parts of blank samples, wherein each 1.00g of blank samples is accurate to +/-0.001 g, putting the blank samples into a 15mL plastic centrifuge tube, respectively adding 0.20mL and 0.50mL of 0.1mg/L hydroxymethylfurfural standard working solution, 0.10mL, 0.20mL and 0.50mL of 1mg/L hydroxymethylfurfural standard working solution, respectively adding 1.5mL of water, adding 1.5mL of ammonia water solution with volume concentration of 3%, fully dissolving until no royal jelly particles are visible to naked eyes, adding 8mL of ethyl acetate, manually oscillating and extracting for 1min, centrifugally separating at the rotating speed of 4500 rpm for 5min, sucking all ethyl acetate layers into a 50mL heart-shaped bottle, rotating in water bath at 40 ℃ to evaporate to dryness, adding 1mL of 6% methanol solution into the heart-shaped flask, ultrasonically dissolving residues, and filtering the sample solution with a 0.22 mu m filter membrane to obtain a series of blank sample standard solutions with the concentrations of 20 mu g/L, 50 mu g/L, 100 mu g/L, 200 mu g/L and 500 mu g/L;
4) and (3) determination:
respectively and precisely sucking 20 mu L of each of 5 parts of series of blank sample standard solutions and test sample solutions, injecting the blank sample standard solutions and the test sample solutions into a liquid chromatograph, drawing a standard working curve by taking the blank sample standard concentration as a horizontal coordinate and the hydroxymethylfurfural peak area as a vertical coordinate, and quantifying by using an external standard method.
In order to verify the stability, accuracy, specificity and system applicability of the method for determining the content of the hydroxymethylfurfural, a methodological verification test is carried out on the method.
Extraction of hydroxymethyl furfural from royal jelly
The extraction method of hydroxymethylfurfural among the existing hydroxymethylfurfural measurement methods is mainly shown in table 1.
TABLE 1 extraction of hydroxymethylfurfural from different samples
As can be seen from table 1 above, for the samples with low protein content, light color and low content of soluble substances, the literature mostly adopts a direct filtration sampling method, for the samples with deep color and high content of soluble substances, ethyl acetate extraction and C18 solid phase extraction cartridges are used for purification, and for the samples with high protein content, a potassium ferrocyanide/zinc acetate protein precipitation method is used for purification.
Royal jelly and royal jelly dry powder contain 15% and 39% of protein, 3% and 10% of fatty acid, and trace pollen pigment, and need to be removed. Therefore, the sample is purified by adopting a potassium ferrocyanide/zinc acetate protein precipitation method, and the hydroxymethyl furfural can be completely extracted, but the detection limit of the method is reduced because the sample is diluted by 20 times, and the detection limit of the method is determined to be 0.3mg/kg, so that the requirement of royal jelly freshness quality control cannot be met.
Second, influence of sample pH on hydroxymethylfurfural extraction
Dissolving a sample with water, extracting with an organic solvent, and concentrating after extracting hydroxymethylfurfural in the sample, thereby improving the detection limit of the method. In practice, we found that the absolute extraction of hydroxymethylfurfural by this process was only 20%. It is possible that the extraction efficiency is affected by the protein in the royal jelly.
The royal jelly is dissolved in alkaline solution, protein is dissolved in alkaline solution, and hydroxymethyl furfural in the royal jelly is extracted by ethyl acetate. We examine the extraction efficiency of ethyl acetate on hydroxymethyl furfural in royal jelly under the condition of alkaline solutions with different concentrations, and the results are shown in Table 2.
TABLE 2 extraction efficiency of hydroxymethylfurfural at different alkaline solution concentrations
As can be seen from table 2, when the pH is neutral, the extraction efficiency of hydroxymethylfurfural from royal jelly is the highest with ethyl acetate. The method can ensure that the protein in the royal jelly is dissolved by the ammonia water, the ethyl acetate and the hydroxymethyl furfural are fully mixed, the sample liquid is in a fine and smooth milk shape, and the contact area of the ethyl acetate and the sample liquid is increased, so that the extraction efficiency is improved. The sample solution without ammonia water can show obvious flocculent protein, and acetic acid and hydroxymethyl furfural cannot be fully mixed, thereby influencing the extraction. When the pH is highly alkaline, the extraction efficiency is inferior to that of a neutral sample solution, which may cause the binding of hydroxymethylfurfural with water, saccharides and proteins in the sample solution to be more firm, and the partition coefficient in ethyl acetate to be small, thereby affecting the extraction efficiency. Therefore, a sample of royal jelly was selected to be dissolved in 1.5mL of water and 1.5mL of 3% aqueous ammonia, and a sample of royal jelly dry powder was selected to be dissolved in 1.5mL of water and 2.0mL of 3% aqueous ammonia. Under different pH conditions, the chromatogram of hydroxymethyl furfural in royal jelly extracted with ethyl acetate is shown in figure 1, wherein the chromatogram is obtained by adding 100 μ g/kg of hydroxymethyl furfural into royal jelly with pH 1 of 3.8, pH 2 of 7.7, pH 3 of 9.6, pH 4 of 7.7 and pH 7.7.
Thirdly, influence of solvent on extraction of hydroxymethyl furfural in royal jelly
Hydroxymethylfurfural is readily soluble in solvents such as water, acetonitrile, acetone, alcohols, ethyl acetate, dimethylformamide, benzene, diethyl ether, chloroform and the like. Alcohol, acetonitrile, acetone and dimethylformamide can be dissolved in water, saccharide in royal jelly is extracted during extraction, and residue obtained after evaporation is large in amount and needs further purification, so that the method is not suitable for use. The ethyl ether is a chemical which is easy to prepare toxin and inconvenient to purchase and manage, and the benzene and the chloroform have certain toxicity, so the ethyl acetate is selected as an extraction solvent.
After water and ammonia water are added into a royal jelly sample, the total volume is about 4.5mL, through tests, when the amount of ethyl acetate is 4mL, the sample liquid is easy to emulsify, when the amount of ethyl acetate is 8mL, the sample liquid is not emulsified, the absolute extraction rate of hydroxymethylfurfural in the sample is more than 60%, when the amount of ethyl acetate is 12mL, the absolute extraction rate of hydroxymethylfurfural in the sample is more than 75%. In consideration of the reagent dosage and the detection sensitivity, the dosage of 8mL ethyl acetate can meet the requirement. Therefore, the amount of ethyl acetate used was selected to be 8 mL.
Selection of mobile phase
We used methanol: 0.4% phosphoric acid 10:90,8: 92 and 6:94 is mobile phase, and chromatographic separation test is carried out. When the ratio of methanol: 0.4% phosphoric acid 10: at 90, an interference peak is arranged before a chromatographic peak of the hydroxymethylfurfural, and when the ratio of methanol: 0.4% phosphoric acid ═ 8: at 92, an interference peak is formed after the chromatographic peak of the hydroxymethylfurfural, although complete separation from the interference peak can be realized in 2 proportions, the impurity peak is relatively close to the chromatographic peak of the hydroxymethylfurfural, when the content of the hydroxymethylfurfural in a sample is high, complete separation of all samples cannot be guaranteed, and when methanol: 0.4% phosphoric acid is 6: at 94 hours, no impurity peak interference exists near the chromatographic peak of the hydroxymethylfurfural, but the retention time of the chromatographic peak of the hydroxymethylfurfural is long, in order to ensure the detection efficiency, the column is washed for 3 minutes by adopting 100 percent methanol after the retention time of the chromatographic peak of the hydroxymethylfurfural, and then the methanol: 0.4% phosphoric acid is 6: stabilizing the chromatographic column for 8min at 94 for 23min, and obtaining a chromatogram after mobile phase optimization, wherein the chromatogram is shown in Table 3 for mobile phase elution conditions and is shown in FIG. 2. Wherein 1, adding 100 mu g/kg hydroxymethylfurfural into the blank sample, 2, adding the blank sample,
TABLE 3 mobile phase gradient elution conditions
Fourthly, the linear range, the recovery rate, the precision and the detection limit of the method
Analytical method
4.1 preparation of test solutions
Weighing 1.00g of royal jelly sample or 0.50g (accurate to +/-0.001 g) of royal jelly dry powder sample into a 15mL plastic centrifuge tube, adding 1.5mL of water, adding 1.5mL of 3% ammonia water solution into the royal jelly sample, adding 2.0mL of 3% ammonia water solution into the royal jelly dry powder sample, fully dissolving until no royal jelly particles can be seen by naked eyes, adding 8mL of ethyl acetate, manually oscillating and extracting for 1min, and centrifuging at the rotating speed of 4500 rpm for 5 min. Sucking all ethyl acetate layer into 50mL heart-shaped flask, rotary evaporating to dryness in water bath at 40 deg.C, adding 1mL 6% methanol solution into the heart-shaped flask, ultrasonic dissolving residue, and filtering the sample solution with 0.22 μm filter membrane to obtain test solution.
4.2 preparation of blank sample Standard solution
Weighing 5 parts of blank samples, namely 1.00g of each blank sample, accurately obtaining +/-0.001 g of each blank sample, adding 0.20mL of 0.50mL of 0.1mg/L hydroxymethylfurfural standard working solution, 0.10mL of 0.20mL of 0.50mL of 1mg/L hydroxymethylfurfural standard working solution and 0.50mL of 1mg/L hydroxymethylfurfural standard working solution into a 15mL plastic centrifuge tube, and carrying out the operation according to the method from 4.1 'adding 1.5mL of water' to obtain the blank sample standard working solution. The concentrations corresponding to the standard solutions were 20. mu.g/L, 50. mu.g/L, 100. mu.g/L, 200. mu.g/L, and 500. mu.g/L.
4.3 liquid chromatography reference conditions
A chromatographic column: c18 packed column (4.6 mm. times.250 mm, 5 μm); or an equivalent chromatographic column.
Mobile phase: mobile phase a, methanol + 0.4% phosphoric acid solution ═ 6+ 94; mobile phase B, methanol. The gradient elution conditions are shown in Table 3.
Flow rate: see table 3.
Column temperature: 35 ℃ is carried out.
Detection wavelength: 285 nm.
Sample introduction amount: 20 μ L.
4.4 determination
Respectively injecting sample into a blank sample standard solution (4.2) and a sample solution (4.1), drawing a standard working curve by taking the concentration of the blank sample standard solution as a horizontal coordinate and the area of the peak of the hydroxymethylfurfural as a vertical coordinate, and quantifying by using an external standard method. The response values of the hydroxymethylfurfural in the blank sample standard solution and the test sample solution are within the linear range detected by the instrument.
Blank matrix samples and samples were processed and measured analytically and working curves were plotted as hydroxymethylfurfural peak area versus mass concentration (x, μ g/kg). As shown in Table 4, the hydroxymethylfurfural showed good linearity in the range of 20. mu.g/kg to 500. mu.g/kg, and the correlation coefficient R 2 0.9983, meets the requirement of quantitative analysis. The detection limit and the quantification limit of the method are respectively 6 mug/kg and 20 mug/kg, the recovery rate is between 82% and 106% (n is 3), and the precision is between 4.6% and 8.0% (n is 3).
When the amount of the sample, water, ammonia water and ethyl acetate is doubled, the extract is dissolved by 1ml of methanol solution after being concentrated for analysis, the detection limit can be increased to 3ug/kg, and the quantification limit is increased to 10ug/kg
TABLE 4 Linear Range, regression equation, correlation coefficient and detection limits for hydroxymethylfurfural
Adding hydroxymethylfurfural standard solutions of 20 mug/kg, 100 mug/kg and 500 mug/kg into blank royal jelly samples respectively, processing according to an analysis method, then carrying out a recovery rate experiment, and repeatedly measuring each addition level for 3 times. As a result, as shown in Table 5, the addition recoveries were 94.8%, 96.7% and 102.9%, respectively, and the relative standard deviations were 5.3%, 4.2% and 2.9%, respectively.
TABLE 5 recovery and precision of hydroxymethylfurfural from the blank Royal jelly
The invention relates to a method for measuring the content of hydroxymethylfurfural in royal jelly and dry powder thereof, which comprises the steps of dissolving and diluting a sample by water and 3% ammonia water, extracting the hydroxymethylfurfural by ethyl acetate, analyzing by using a high performance liquid chromatograph, and quantifying by using an external standard method. The method has the detection limit of 6 mug/kg, the quantitative limit of 20 mug/kg, the recovery rate of 82-106% (n is 3) and the precision of 4.6-8.0% (n is 3), and can meet the judgment requirement on the freshness of the royal jelly and the royal jelly dry powder.
The royal jelly dry powder is a product obtained by dehydrating royal jelly, the content of the hydroxymethylfurfural of the royal jelly is far higher than that of the royal jelly, and the judgment of the freshness of the royal jelly dry powder by using the content of the hydroxymethylfurfural is more meaningful than that of the royal jelly.
Drawings
FIG. 1 is a chromatogram of hydroxymethyl furfural in royal jelly extracted with ethyl acetate under different pH conditions;
FIG. 2 is a chromatogram of hydroxymethylfurfural in a sample after mobile phase optimization.
Detailed Description
The present invention is further described below with reference to examples.
Example 1
Method for detecting content of hydroxymethyl furfural in royal jelly and dry powder thereof
Instruments and reagents
The Shimadzu LC-20A high performance liquid chromatograph is provided with an LC-20AVP ultraviolet detector; sartorius BS224S electronic analytical balance: the sensory quantity is 0.0001 g; KQ3200E ultrasonic cleaner, ultrasonic instruments ltd, kunshan; SC-3612 desk type high speed centrifuge of Zhongjia scientific instruments ltd, Anhui; and (3) standard substance: hydroxymethylfurfural standard, CAS: 67-47-0 and the purity is more than 99 percent. Phosphoric acid: 85 percent and premium grade purity; methanol, pure liquid chromatography; 28-30% of ammonia water; the ethyl acetate is analytically pure; water, which conforms to GB/T6682 first-grade water. Filter, nylon 66, 0.22 μm.
3% ammonia solution: 3.0mL of ammonia water is sucked and the volume is adjusted to 100 mL.
6% methanol solution: 6.0mL of methanol was taken and the volume was adjusted to 100mL with water.
0.4% phosphoric acid solution: 4.0mL of phosphoric acid was taken up and made up to 1000mL with water.
Standard stock solution of hydroxymethylfurfural: weighing 20mg of hydroxymethylfurfural standard, adding methanol to a constant volume of 20mL to prepare 1.0mg/mL hydroxymethylfurfural stock solution. Store at-18 ℃ for 24 months.
Standard intermediate solution of hydroxymethylfurfural: sucking 0.1mL of hydroxymethylfurfural stock solution, adding water to a constant volume of 10mL to prepare 10mg/L of hydroxymethylfurfural standard intermediate solution, and storing at-18 ℃ for 12 months.
Standard hydroxymethylfurfural working solution i: sucking 1.0mL of hydroxymethylfurfural standard intermediate solution, adding water to a constant volume of 10mL to prepare 1mg/L hydroxymethylfurfural standard working solution I, and storing at-18 ℃ for 12 months.
Hydroxymethyl furfural standard working solution ii: sucking 1.0mL of hydroxymethylfurfural standard working solution I, and adding water to a constant volume of 10mL to prepare 0.1mg/L of hydroxymethylfurfural standard working solution II, and storing at-18 ℃ for 12 months.
The royal jelly and royal jelly dry powder samples are provided by Hangzhou Biyuntian health products Co.
1) Determining chromatographic conditions:
chromatographic column packing agent: octadecyl bonded silica gel;
mobile phase: mobile phase A, methanol-0.4% phosphoric acid solution with the volume ratio of 6: 94; mobile phase B, methanol; detection wavelength: 285 nm; column temperature: 35 ℃;
2) preparation of a test solution:
weighing 1.00g of royal jelly sample or 0.50g of royal jelly dry powder sample, accurately obtaining the sample to +/-0.001 g, adding 1.5mL of water into a 15mL plastic centrifuge tube, adding 1.5mL of 3% ammonia water solution with volume concentration into the royal jelly sample, adding 2.0mL of 3% ammonia water solution with volume concentration into the royal jelly dry powder sample, fully dissolving until royal jelly particles can not be seen by naked eyes, adding 8mL of ethyl acetate, manually oscillating and extracting for 1min, and centrifugally separating at the rotating speed of 4500 rpm for 5 min; sucking all ethyl acetate layers into a 50mL heart-shaped flask, rotating in a water bath at 40 ℃ to evaporate to dryness, adding 1mL methanol solution with the concentration of 6% into the heart-shaped flask, dissolving residues by ultrasonic waves, and filtering the sample solution with a 0.22 mu m filter membrane to obtain 1.00g of sample solution per 1 mL;
3) preparation of blank sample standard solution:
weighing 5 parts of blank samples, namely 1.00g of each blank sample, accurately obtaining +/-0.001 g of each blank sample, adding 0.20mL of hydroxymethylfurfural standard working solution II, 0.50mL of hydroxymethylfurfural standard working solution II, 0.10mL of hydroxymethylfurfural standard working solution I, 0.20mL of hydroxymethylfurfural standard working solution I and 0.50mL of hydroxymethylfurfural standard working solution I into a 15mL plastic centrifuge tube, adding 1.5mL of water, adding 1.5mL of ammonia water solution with the volume concentration of 3%, fully dissolving the mixture until royal jelly particles can not be seen by naked eyes, adding 8mL of ethyl acetate, manually shaking and extracting the mixture for 1min, and centrifugally separating the mixture for 5min at the rotating speed of 4500 rpm. Sucking all ethyl acetate layers into a 50mL heart-shaped flask, carrying out rotary evaporation in a water bath at 40 ℃ to dryness, adding 1mL 6% methanol solution into the heart-shaped flask, ultrasonically dissolving residues, and filtering the sample solution through a 0.22 mu m filter membrane to obtain control solutions with the concentrations of 20 mu g/L, 50 mu g/L, 100 mu g/L, 200 mu g/L and 500 mu g/L;
4) and (3) determination:
respectively and precisely sucking 5 parts of reference solution and 20 mu L of test solution respectively, injecting into a liquid chromatograph, drawing a reference solution curve by taking the concentration of the reference solution as a horizontal coordinate and the area of the peak of the hydroxymethylfurfural as a vertical coordinate, and quantifying the sample by using the reference solution curve.
11 samples of royal jelly were measured by the method of example, and the results are shown in Table 6.
TABLE 6 measurement results of royal jelly and royal jelly dry powder samples
From the detection situation of the actual samples, the content of the hydroxymethylfurfural in the new royal jelly of 2022 years is below 12 mu g/kg when the new royal jelly is stored at the temperature of 18 ℃ below zero, the content of the hydroxymethylfurfural in the royal jelly of 2021 years is below 33 mu g/kg when the royal jelly is stored at the temperature of 18 ℃ below zero, and the content of the hydroxymethylfurfural in the royal jelly stored at the temperature of 4 ℃ is between 91 and 280 mu g/kg. The correlation between the content of the hydroxymethylfurfural in the royal jelly and the storage temperature and time is proved.
The aqueous ammonia may be replaced by an aqueous carbonate solution, an aqueous sodium hydroxide solution or an aqueous calcium hydroxide solution according to the method of example 1, as long as the pH of the sample solution is 7 to 8,
example 2
The procedure of example 1 was followed except that mobile phase A was a 0.4% phosphoric acid solution.
Example 3
The procedure of example 1 was followed except that the mobile phase A was methanol-0.4% phosphoric acid solution at a stripping ratio of 10: 90.
Example 4
The procedure is as in example 1 except that the mobile phase A, methanol-0.4% phosphoric acid solution, is used in a 2:98 stripping ratio.
Furthermore, it should be noted that equivalent or simple changes may be made in the structure, features and principles described in the present patent concepts, such as: according to the preparation method of the test solution, the obtained test solution adopts other instruments to perform qualitative or quantitative analysis on the furfural content; changes in the amount of sample, changes in the volume of ammonia and ethyl acetate, etc., are included in the scope of the present invention.
Claims (5)
1. The method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof is characterized by adopting a high performance liquid chromatography and an external standard method for quantification, and comprises the following steps:
1) determining chromatographic conditions:
chromatographic column packing agent: octadecyl bonded silica gel;
mobile phase: the mobile phase A is methanol-0.4% phosphoric acid solution, and the volume ratio is 0-10: 90-100; mobile phase B, methanol; detection wavelength: 285 nm; column temperature: 35 ℃;
2) preparation of a test solution:
weighing a royal jelly sample or a royal jelly dry powder sample, dissolving in an alkaline solution, extracting with ethyl acetate, evaporating an ethyl acetate layer to dryness, adding a methanol solution, and filtering with a filter membrane to obtain a sample solution containing 1.00g of sample per 1 ml;
3) preparation of blank sample standard solution:
weighing 5 parts of blank sample, adding hydroxymethylfurfural standard working solution to obtain blank sample standard solution with the concentration of 20 mug/L, 50 mug/L, 100 mug/L, 200 mug/L and 500 mug/L;
4) and (3) determination:
respectively and precisely absorbing 20 mu L of blank sample standard solution and sample solution, injecting the blank sample standard solution and the sample solution into a liquid chromatograph, drawing a standard working curve by taking the concentration of the blank sample standard solution as a horizontal coordinate and the peak area of the hydroxymethylfurfural as a vertical coordinate, and quantifying by an external standard method.
2. The method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof according to claim 1, wherein the mobile phase A is a methanol-0.4% phosphoric acid solution, and the volume ratio is 6: 94.
3. The method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof according to claim 1, wherein the alkaline solution is selected from ammonia, an aqueous carbonate solution, an aqueous sodium hydroxide solution or an aqueous calcium hydroxide solution, so that the pH of a sample solution is 7-8.
4. The method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof according to claim 1, wherein the detection limit of the method is 3-6 μ g/kg, and the quantification limit is 10-20 μ g/kg.
5. The method for detecting the content of hydroxymethylfurfural in royal jelly and dry powder thereof is characterized by adopting a high performance liquid chromatography and an external standard method for quantification, and comprises the following steps:
1) determining chromatographic conditions:
chromatographic column packing agent: octadecyl bonded silica gel;
mobile phase: mobile phase A, methanol-0.4% phosphoric acid solution with the volume ratio of 6: 94; mobile phase B, methanol; detection wavelength: 285 nm; column temperature: 35 ℃;
2) preparation of a test solution:
weighing 1.00g of royal jelly sample or 0.50g of royal jelly dry powder sample, accurately obtaining the sample to +/-0.001 g, adding 1.5mL of water into a 15mL plastic centrifuge tube, adding 1.5mL of 3% ammonia water solution with volume concentration into the royal jelly sample, adding 2.0mL of 3% ammonia water solution with volume concentration into the royal jelly dry powder sample, fully dissolving until royal jelly particles can not be seen by naked eyes, adding 8mL of ethyl acetate, manually oscillating and extracting for 1min, and centrifugally separating at the rotating speed of 4500 rpm for 5 min; sucking all ethyl acetate layers into a 50mL heart-shaped flask, rotating in a water bath at 40 ℃ to evaporate to dryness, adding 1mL of 6% methanol solution into the heart-shaped flask, ultrasonically dissolving residues, and filtering the sample solution with a 0.22 mu m filter membrane to obtain 1.00g of test solution per 1 mL;
3) preparation of blank sample standard solution:
weighing 5 parts of blank samples, wherein each 1.00g of blank samples is accurate to +/-0.001 g, putting the blank samples into a 15mL plastic centrifuge tube, respectively adding 0.20mL and 0.50mL of 0.1mg/L hydroxymethylfurfural standard working solution, 0.10mL, 0.20mL and 0.50mL of 1mg/L hydroxymethylfurfural standard working solution, respectively adding 1.5mL of water, adding 1.5mL of ammonia water solution with volume concentration of 3%, fully dissolving until no royal jelly particles are visible to naked eyes, adding 8mL of ethyl acetate, manually oscillating and extracting for 1min, centrifugally separating at the rotating speed of 4500 rpm for 5min, sucking all ethyl acetate layers into a 50mL heart-shaped bottle, rotating in water bath at 40 ℃ to evaporate to dryness, adding 1mL of 6% methanol solution into the heart-shaped bottle, ultrasonically dissolving residues, and filtering the sample solution with a 0.22 mu m filter membrane to obtain blank sample standard solutions with the concentrations of 20 mu g/L, 50 mu g/L, 100 mu g/L, 200 mu g/L and 500 mu g/L;
4) and (3) determination:
respectively and precisely sucking 5 parts of blank sample standard solution and test solution by 20 mu L, injecting into a liquid chromatograph, drawing a standard working curve by taking the concentration of the blank sample standard solution as a horizontal coordinate and the area of the peak of the hydroxymethylfurfural as a vertical coordinate, and quantifying by an external standard method.
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