CN112505165A - Method for measuring residual quantity of dicyandiamide in dairy product - Google Patents
Method for measuring residual quantity of dicyandiamide in dairy product Download PDFInfo
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- CN112505165A CN112505165A CN202010666979.1A CN202010666979A CN112505165A CN 112505165 A CN112505165 A CN 112505165A CN 202010666979 A CN202010666979 A CN 202010666979A CN 112505165 A CN112505165 A CN 112505165A
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- dicyandiamide
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- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 title claims abstract description 146
- 235000013365 dairy product Nutrition 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 54
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 170
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000012224 working solution Substances 0.000 claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 22
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 20
- 238000000746 purification Methods 0.000 claims abstract description 15
- 239000011159 matrix material Substances 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 6
- 239000006185 dispersion Substances 0.000 claims abstract description 5
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 63
- 150000002500 ions Chemical class 0.000 claims description 40
- -1 n-hexane saturated acetonitrile Chemical class 0.000 claims description 31
- 239000006228 supernatant Substances 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 20
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- VLKZOEOYAKHREP-UHFFFAOYSA-N methyl pentane Natural products CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 14
- 239000012071 phase Substances 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000011550 stock solution Substances 0.000 claims description 12
- 239000007789 gas Substances 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims description 10
- 238000007865 diluting Methods 0.000 claims description 8
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 4
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 238000005341 cation exchange Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000004807 desolvation Methods 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 2
- 239000000843 powder Substances 0.000 description 26
- 235000013336 milk Nutrition 0.000 description 23
- 239000008267 milk Substances 0.000 description 23
- 210000004080 milk Anatomy 0.000 description 23
- 238000001819 mass spectrum Methods 0.000 description 20
- 238000011084 recovery Methods 0.000 description 11
- 229920000877 Melamine resin Polymers 0.000 description 10
- 239000012496 blank sample Substances 0.000 description 10
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 10
- 238000001514 detection method Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 5
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- 239000000047 product Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000005374 Poisoning Diseases 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 241000108664 Nitrobacteria Species 0.000 description 1
- 206010038743 Restlessness Diseases 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- CFNHVUGPXZUTRR-UHFFFAOYSA-N n'-propylethane-1,2-diamine Chemical compound CCCNCCN CFNHVUGPXZUTRR-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Images
Classifications
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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
-
- 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
- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- 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
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention provides a method for measuring the residual amount of dicyandiamide in a dairy product, which is characterized by comprising the following steps: s1: preparing dicyandiamide standard working solutions with different concentrations; s2: ultrasonically extracting residual dicyandiamide in the dairy product by using a mixed solution of acetonitrile and water, and performing matrix dispersion solid phase extraction and purification to prepare a liquid to be detected; s3: performing mass spectrometry on the dicyandiamide standard working solution prepared in the step S1 by using a liquid chromatography tandem mass spectrometer, and drawing a standard curve; s4: and (4) performing mass spectrometry on the liquid to be detected in the step S2 by using a liquid chromatography tandem mass spectrometer, and obtaining the content of the dicyandiamide in the dairy product through the standard curve of the step S3. The determination method can be used for independently, simply and accurately determining the residual amount of dicyandiamide in the dairy product.
Description
Technical Field
The invention belongs to the technical field of food detection and chemical analysis, and relates to a method for determining residual quantity of dicyandiamide in a dairy product.
Background
Dicyandiamide, also known as dicyanodiamide, abbreviated as DICY or DCD, has the chemical formula C2H4N4. Dicyandiamide is an industrial raw material and is often used as an additive of a compound fertilizer to control the activity of nitrobacteria, adjust the conversion rate of a nitrogen fertilizer in soil, reduce the loss of ammonium nitrogen and achieve the purpose of improving the use efficiency of the fertilizer.
In 2013, in 1 month, dicyandiamide is contained in part of milk powder produced by a huge natural group of the New Zealand dairy industry, and the reason is that dicyandiamide residues are generated in the milk powder because a chemical fertilizer containing dicyandiamide is used by New Zealand dairy farmers. Dicyandiamide is a raw material for producing melamine, also belongs to a non-protein high-nitrogen substance, and the safety of dicyandiamide is not evaluated. In addition, dicyandiamide, as a dimer of dicyandiamide, has similar poisoning symptoms to those of dicyandiamide trimer melamine in mice, and mice administered with 13000mg/kg doses of dicyandiamide and melamine have consistent poisoning symptoms, all of which are restless, jumpy and rapid in respiration, and then die within tens of minutes. Given that it may be as harmful as melamine to infants and young children, the new zealand government banned the sale of dairy products containing dicyandiamide. China is the main import country of the milk powder in New Zealand, about 40 percent of the milk powder in the market is from New Zealand at present, and the incident again arouses the high concern and question of people on the safety of the milk powder.
Chinese patent CN102419354A discloses a general rapid detection method for small molecule harmful substances in liquid milk, and particularly discloses a method for measuring the small molecule harmful substances in the liquid milk by combining ultra-high performance liquid chromatography and triple quadrupole mass spectrometry; the method is suitable for detecting micromolecular harmful substances such as melamine, dicyandiamide and the like in the liquid milk.
Chinese patent CN102435699A discloses a method for rapidly detecting melamine in milk and a milk product by liquid chromatography-tandem mass spectrometry, and specifically discloses steps of adding an internal standard into a milk product to be detected, extracting melamine by acetonitrile, and qualitatively and quantitatively analyzing and measuring the melamine in the milk product by using a normal phase chromatographic column-tandem mass spectrometry MRM (mass spectrometry multiple reaction monitoring).
Chinese patent CN103217498A discloses a method for detecting dicyandiamide in milk powder by liquid chromatography-mass spectrometry and a sample pretreatment method, wherein the method comprises the following steps: sample pretreatment: the sample was dissolved in water and extracted by adding organic extract, followed by MgSO4And NaCl enables the organic extracting solution and the water phase to be layered, a first supernatant is obtained through centrifugation, an adsorbent is added into the first supernatant for purification, a second supernatant is obtained through centrifugation, and the second supernatant is taken to be concentrated, redissolved and filtered to obtain a sample to be detected; and then, detecting dicyandiamide contained in the milk powder by using an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer. The process of preparing the sample to be tested in the method is complicated, time-consuming and labor-consuming, and the addition of NaCl can cause salting-out effect and reduce the solubility of the sample, thereby causing inaccurate test results.
Disclosure of Invention
Aiming at part or all of the technical problems in the prior art, the invention provides a method for measuring the residual amount of dicyandiamide in a dairy product, which can more simply, conveniently and accurately measure the residual amount of dicyandiamide in the dairy product.
In order to achieve the aim, the invention provides a method for measuring the residual quantity of dicyandiamide in a dairy product, which comprises the following steps:
s1: preparing dicyandiamide standard working solutions with different concentrations;
s2: ultrasonically extracting residual dicyandiamide in the dairy product by using a mixed solution of acetonitrile and water, and performing matrix dispersion solid phase extraction and purification to prepare a liquid to be detected;
s3: performing mass spectrometry on the dicyandiamide standard working solution prepared in the step S1 by using a liquid chromatography tandem mass spectrometer, and drawing a standard curve;
s4: and (4) performing mass spectrometry on the liquid to be detected in the step S2 by using a liquid chromatography tandem mass spectrometer, and obtaining the content of the dicyandiamide in the dairy product through the standard curve of the step S3.
In one embodiment, the step of configuring the dicyandiamide standard working solution in the step S1 includes the following steps:
s11: taking a proper amount of dicyandiamide internal standard substance, and preparing a standard stock solution with the concentration of not less than 100 mug/mL by using pure water;
s12: diluting the stock solution into a standard intermediate solution with 10 mug/mL by using a pure water solution;
s13: and diluting the intermediate solution with water to obtain standard working solution with various concentrations.
In one embodiment, the extraction of residual dicyandiamide in the dairy product in step S2 includes the following steps:
s21: weighing a sample, adding water, performing ultrasonic treatment, adding n-hexane saturated acetonitrile and acetonitrile saturated n-hexane, performing vortex mixing uniformly, oscillating, centrifuging, and extracting an acetonitrile layer.
In one embodiment, the extraction of residual dicyandiamide in the dairy product in step S2 includes the following steps:
weighing 1g-5g of sample, adding 2mL-10mL of water, performing ultrasonic treatment for 5min-15min, adding 10mL-30mL of n-hexane saturated acetonitrile and 5mL-15mL of acetonitrile saturated n-hexane, performing vortex mixing uniformly, performing extraction for 10min-30min by oscillation, and centrifuging for 2min-8min at 10000r/min-18000 r/min; and extracting the acetonitrile layer.
In one embodiment, the purging of the acetonitrile layer in step S2 includes the steps of:
and (4) adding the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with anhydrous magnesium sulfate, C18, PSA and GCB, uniformly mixing by vortex, oscillating, centrifuging, taking the supernatant into a small centrifuge tube, centrifuging again, and taking the supernatant to be tested.
In one embodiment, the purging of the acetonitrile layer in step S2 includes the steps of:
adding 1-5 mL of the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with 200-600 mg of anhydrous magnesium sulfate, 60-150 mg of C18, 80-150 mg of PSA and 30-100 mg of GCB, uniformly mixing by vortex, oscillating for 8-15 min, centrifuging for 1-10 min at 10000-18000 r/min, taking 0.8-2 mL of supernatant to a small centrifuge tube, centrifuging for 1-10 min at 10000-18000 r/min, taking the supernatant and bottling to be tested.
In one embodiment, obtaining the content of dicyandiamide according to the standard curve of step S3 in step S4 includes:
s41: determining the liquid to be detected in the step S2 by adopting a liquid chromatography tandem mass spectrometer to obtain the peak area of the liquid to be detected;
s42: the concentration c of the component to be measured in the sample solution is obtained by checking a standard curve according to the peak area;
s43: according to the formula
Calculating the content of dicyandiamide in the sample, wherein c represents the concentration of the component to be detected in the sample solution, ng/mL, which is obtained by looking up a standard curve; m represents the sample mass, g; x represents the dicyandiamide content in the sample, mg/kg.
In one embodiment, the assay method further comprises a blank test, and the assay of the blank test comprises:
and selecting a negative sample, and performing mass spectrometry by adopting a liquid chromatography tandem mass spectrometer according to the step of measuring the standard working solution to obtain a mass spectrogram of a blank test.
In one embodiment, the chromatographic conditions employed in steps S3 and S4 comprise:
a) a chromatographic column: waters BEH Amide, 1.7 μm × 2.1mm × 50 mm;
b) temperature of the column: 30 ℃;
c) mobile phase: (5mmol/L amine acetate + 0.1% formic acid)/acetonitrile (5/95);
d) flow rate: 0.2 mL/min;
e) sample introduction amount: 2.5. mu.L.
In one embodiment, the chromatographic conditions employed in steps S3 and S4 comprise:
a2) liquid chromatography column: cation exchange and C18 mixed column CR (1: 4) 2.0X 150mm X5 μm;
b2) column temperature: 35 ℃;
c2) mobile phase: mobile phase C (20mmol/L ammonium acetate + 0.2% formic acid) to mobile phase B (acetonitrile) 90: 10;
d2) flow rate: 0.3 mL/min;
e2) sample introduction amount: 5 μ L.
In one embodiment, the mass spectrometry conditions employed in steps S3 and S4 include:
an ionization mode: ESI +;
taper hole airflow: nitrogen gas, 40L/h;
capillary voltage: 3.0 kV;
desolventizing the gas stream: nitrogen gas, 1000L/h;
source temperature: 150 ℃;
collision gas: argon gas;
the temperature of the desolvation: 500 ℃;
and (3) monitoring mode: multiple Reaction Monitoring (MRM).
In one embodiment, the multi-reaction monitoring ion pairs, collisional gas energy, cone hole voltage, residence time employed in steps S3 and S4 includes:
dicyandiamide compound, parent ion 84.8 and daughter ion 67.75 are adopted; the adopted taper hole voltage is 10V, and the collision energy is 13 eV;
adopting dicyandiamide compound, wherein the cone hole voltage adopted by the parent ion 84.8 and the daughter ion 45.85 is 13V, and the collision energy is 13 eV;
adopting dicyandiamide internal standard compound, mother ion 88.8 and daughter ion 70.75; the cone hole voltage is 25V, and the collision energy is 13 eV.
Compared with the prior art, the invention has the advantages that:
in the invention, the dicyandiamide remained in the sample is extracted by ultrasonic through a mixed solution of acetonitrile and water, the matrix is extracted and purified, the ultra-high performance liquid chromatography tandem mass spectrometry is adopted for determination, and the internal standard method is used for quantification. Compared with the prior art, the method can independently and accurately measure the residual dicyandiamide content in the sample. The determination method is simpler and more convenient, and has sensitive determination and high recovery rate.
Drawings
Preferred embodiments of the present invention will be described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a standard quality spectrum of dicyandiamide at 0.01. mu.g/mL;
FIG. 2 is a mass spectrum of a blank sample of milk powder in example one;
FIG. 3 is a blank sample plus standard mass spectrum of milk powder in example one;
FIG. 4 is a standard operating curve for dicyandiamide of example one;
FIG. 5 is a mass spectrum of a standard solution of dicyandiamide 0.01. mu.g/mL in example two;
FIG. 6 is a mass spectrum of a sample of defatted flour of example two;
FIG. 7 is a mass spectrum of a blank sample of the defatted powder of example two;
FIG. 8 is a mass spectrum of the sample of the defatted powder of example two with the addition of the recovered sample.
In the drawings, like parts are provided with like reference numerals. The figures are not drawn to scale.
Detailed Description
In order to make the technical solutions and advantages of the present invention more apparent, exemplary embodiments of the present invention are described in further detail below with reference to the accompanying drawings. It is clear that the described embodiments are only a part of the embodiments of the invention, and not an exhaustive list of all embodiments. And the embodiments and features of the embodiments may be combined with each other without conflict.
The inventor notices in the process of invention that most of the current determination of dairy products are directed to both melamine and dicyandiamide, and the measurement result is easy to be inaccurate when the dicyandiamide is measured by using the method due to the close performance of the melamine and the dicyandiamide. In the method for separately measuring dicyandiamide, the process of preparing the sample to be measured is complex, time and labor are wasted, and the addition of NaCl can cause salting-out effect, reduce the solubility of the sample and further cause inaccurate test results.
In view of the above disadvantages, the embodiment of the present invention provides a method for measuring the residual amount of dicyandiamide in a dairy product, which is described in detail below.
In one embodiment, the reagents and materials required to be prepared in the method for determining the residual amount of dicyandiamide in the dairy product of the invention comprise:
1) aqueous solution: the water is ultrapure water (for laboratory use);
2) n-hexane (chromatographically pure);
3) acetonitrile (chromatographically pure);
4) amine acetate (guaranteed reagent);
5) anhydrous magnesium sulfate (analytical grade);
6) graphitized carbon as an adsorbent;
7) adsorbent PSA (N-propylethylenediamine);
8) adsorbent C18;
9) dicyandiamide standard substance: english name: a dicyclandiamide; CAS: 461-581-5; relative molecular mass: 84.08, respectively; the molecular formula is as follows: c2H4N4；
10) Dicyandiamide isotope internal standard: english name: dicyandiamide-15N 4; relative molecular mass: 87.7.
in one embodiment, the method for determining the residual amount of dicyandiamide in the dairy product of the present invention includes the steps of:
11) ultra performance liquid chromatography tandem mass spectrometry: an ESI source (Electron Spray Ionization);
12) a vortex mixer;
13) an analytical balance;
14) an oscillator;
15)50mL high-speed centrifuge tube;
16)50mL of low-speed centrifuge tube;
17)5mL centrifuge tube; 2mL of small centrifuge tube;
18) a volumetric flask;
19) a pipette.
The first embodiment is as follows:
in this example, the sample used was a certain brand of milk powder. The method for determining the residual amount of dicyandiamide in the dairy product mainly comprises the following steps:
s1: preparing dicyandiamide standard working solutions with different concentrations;
s2: performing ultrasonic extraction on residual dicyandiamide in a dairy product (milk powder of a certain brand in the embodiment) by using a mixed solution of acetonitrile and water, and performing matrix dispersion solid-phase extraction and purification to prepare a to-be-detected liquid;
s3: performing mass spectrometry on the dicyandiamide standard working solution prepared in the step S1 by using a liquid chromatography tandem mass spectrometer, and drawing a standard curve;
s4: and (4) performing mass spectrometry on the liquid to be detected in the step S2 by using a liquid chromatography tandem mass spectrometer, and obtaining the content of the dicyandiamide in the dairy product through the standard curve of the step S3.
In this embodiment, the method for determining the residual amount of dicyandiamide in the dairy product of the present invention includes the preparation of a standard working solution of dicyandiamide. More specifically, the preparation of dicyandiamide standard working solution includes:
s11, preparing dicyandiamide internal standard stock solution: accurately weighing a proper amount of dicyandiamide internal standard (accurate to 0.1mg), and preparing a standard stock solution with the concentration of not less than 100 mug/mL (the standard stock solution can be stored for 1 year at 4 ℃) by using water;
s12, preparing dicyandiamide internal standard intermediate liquid: diluting the internal standard stock solution into a standard intermediate solution of 10 mug/mL;
s13, preparing dicyandiamide standard working solution: and diluting the standard intermediate solution of the dicyandiamide with water into standard working solution with the concentration of 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL and 50 ng/mL.
Preferably, the method for determining the residual amount of dicyandiamide in the dairy product further comprises the following steps of sample preparation and preservation: and (3) putting about 200g of a sample into a clean container as a sample, sealing and marking, and storing at normal temperature.
Further, the step S2 of the method for determining the residual amount of dicyandiamide in the dairy product of the present invention for extracting the residual dicyandiamide in the dairy product includes the following steps:
s21: weighing a sample, adding water, performing ultrasonic treatment, adding n-hexane saturated acetonitrile and acetonitrile saturated n-hexane, performing vortex mixing uniformly, oscillating, centrifuging, and extracting an acetonitrile layer.
Preferably, in this embodiment, the step S2 of extracting the residual dicyandiamide in the dairy product includes the following steps:
weighing 1g-5g of sample, adding 2mL-10mL of water, performing ultrasonic treatment for 5min-15min, adding 10mL-30mL of n-hexane saturated acetonitrile and 5mL-15mL of acetonitrile saturated n-hexane, performing vortex mixing uniformly, performing extraction for 10min-30min by oscillation, and centrifuging for 2min-8min at 10000r/min-18000 r/min; and extracting the acetonitrile layer.
In a preferred embodiment, the extraction of residual dicyandiamide in the dairy product in step S2 mainly comprises the following steps:
weighing about 2g of sample (accurate to 0.001g), adding 20 mu L of internal standard, adding 3mL of water, performing ultrasonic treatment for 10min, adding 17mL of n-hexane saturated acetonitrile, performing vortex mixing on 10mL of acetonitrile saturated n-hexane uniformly, performing oscillation extraction for 20min, and centrifuging for 3min at 15000 r/min; and extracting the acetonitrile layer.
In this embodiment, the purification of the acetonitrile layer in step S2 mainly includes the following steps:
and (4) adding the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with anhydrous magnesium sulfate, C18, PSA and GCB, uniformly mixing by vortex, oscillating, centrifuging, taking the supernatant into a small centrifuge tube, centrifuging again, and taking the supernatant to be tested.
In a preferred embodiment, the purification of the acetonitrile layer in step S2 comprises the steps of:
adding 1-5 mL of the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with 200-600 mg of anhydrous magnesium sulfate, 60-150 mg of C18, 80-150 mg of PSA and 30-100 mg of GCB, uniformly mixing by vortex, oscillating for 8-15 min, centrifuging for 1-10 min at 10000-18000 r/min, taking 0.8-2 mL of supernatant to a small centrifuge tube, centrifuging for 1-10 min at 10000-18000 r/min, taking the supernatant and bottling to be tested.
In a further preferred embodiment, the purification of the acetonitrile layer in step S2 comprises the steps of:
weighing 400mg of anhydrous magnesium sulfate, 100mgC18 mg of 100mg of PSA and 50mg of GCB respectively, putting the anhydrous magnesium sulfate, 100mgC18 mg of PSA and 50mg of GCB into a 5mL centrifuge tube, adding 2.5mL of acetonitrile layer obtained by extraction into the 5mL centrifuge tube, uniformly mixing by vortex, oscillating for 10min, centrifuging for 3min at 12000r/min, adding 1.0mL of supernatant into a 2mL small centrifuge tube, centrifuging for 3min at 12000r/min, taking the supernatant, bottling, and measuring by a computer.
Further, the method for determining the residual amount of dicyandiamide in the dairy product comprises the following UPLC chromatographic conditions:
a1) a chromatographic column: waters BEH Amide, 1.7 μm × 2.1mm × 50 mm;
b1) column temperature (i.e. column temperature): 30 ℃;
c1) mobile phase: (5mmol/L amine acetate + 0.1% formic acid)/acetonitrile (5/95);
d1) flow rate: 0.2 mL/min;
e1) sample introduction amount: 2.5. mu.L.
Preferably, the method for measuring the residual amount of dicyandiamide in the dairy product of the invention adopts the mass spectrum conditions shown in the following table 1:
TABLE 1 Mass Spectrometry conditions
As can be seen from the mass spectrum conditions in table 1 above in conjunction with the mass spectrograms of fig. 1 to 4, in this example, the ideal dicyandiamide analysis parameters were obtained by optimizing the parameters such as capillary voltage, ion source temperature, taper hole and desolvation gas flow.
Further, the method for determining the residual amount of dicyandiamide in the dairy product comprises the following determination working conditions of table 2:
TABLE 2 Multi-reaction monitoring of ion-pairs, collisional gas energy, taper hole voltage, dwell time
As can be seen from the measurement conditions in table 2 above and the mass spectrograms in fig. 1 to 4, in this embodiment, the parent ions, the daughter ions, and the collision energies of the dicyandiamide and the internal standard thereof obtained by optimization all achieve ideal effects in the aspects of sensitivity, signal-to-noise ratio, selectivity, and the like.
Further, the method for determining the residual amount of dicyandiamide in the dairy product comprises the following steps:
41) and (4) mass spectrometry. The method comprises the step of respectively measuring a sample to be measured and a standard working solution by using a liquid chromatography tandem mass spectrometer. And (4) drawing a standard curve by taking the concentration of the dicyandiamide in the standard working solution, the concentration of the internal standard and the corresponding peak area ratio as a vertical coordinate.
In one embodiment, the method for determining the residual amount of dicyandiamide in the dairy product further comprises the following steps:
42) and (5) blank testing. Selecting a negative sample, and performing the mass spectrometry step of 41) above.
In one embodiment, the method for determining the residual amount of dicyandiamide in the dairy product comprises the following steps:
43) the content of dicyandiamide in the sample is calculated according to the formula:
in the formula: x represents the content of dicyandiamide in the sample, mg/kg;
c represents the concentration of the component to be measured in the sample solution, ng/mL, as measured from the standard curve;
m represents the sample mass, g;
note: blank values need to be deducted from the calculation result.
Further, fig. 1 to 4 show the mass spectra of the first embodiment. Wherein, FIG. 1 shows the standard quality spectrum of dicyandiamide at 0.01. mu.g/mL. This experiment was performed by mass spectrometry, and two graphs are shown in FIG. 1 showing the mass spectra of different parent ion-daughter ion combinations, where the top graph is the mass spectrum of the parent ion 84.8, daughter ion 67.75 combination, and the bottom graph is the mass spectrum of the parent ion 84.8, daughter ion 45.8 combination. In the mass spectrum shown in FIG. 1, the upper panel shows a retention time of 1.35min and a quantitative ion signal-to-noise ratio > 10. The lower graph shows retention time of 1.36min, qualitative ion signal to noise ratio > 10.
FIG. 2 is a mass spectrum of a blank sample of milk powder; the upper graph is the mass spectrum of the combination of the parent ion 84.8 and the daughter ion 67.75, and the lower graph is the mass spectrum of the combination of the parent ion 84.8 and the daughter ion 45.8. In the mass spectrum, dicyandiamide in the blank sample is not detected and no other interference exists. .
FIG. 3 is a schematic diagram of a blank sample of milk powder with a standard mass spectrum; the upper graph is the mass spectrum of the combination of the parent ion 84.8 and the daughter ion 67.75, and the lower graph is the mass spectrum of the combination of the parent ion 84.8 and the daughter ion 45.8. In the mass spectrogram, the upper graph shows that the retention time of dicyandiamide of the combination of the parent ion 84.8 and the daughter ion 67.75 is 1.36min, the signal-to-noise ratio of the quantitative ion is more than 10, and the recovery rate is 80-110% to meet the quality control requirement without other interference. The following graph shows that the retention time of the combination of the parent ion 84.8 and the daughter ion 45.8 is 1.35min, the signal-to-noise ratio of the quantitative ion is more than 10, and the recovery rate is between 80 and 110 percent to meet the quality control requirement.
Figure 4 is a standard operating curve for dicyandiamide of example one. In the standard working curve, the instrument method of the patent is shown to have good linearity under 6 concentration gradients of 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL and 50 ng/mL.
In this example, the measurement limit of dicyandiamide was 0.05 mg/kg.
In this example, the added concentration and recovery rate range of dicyandiamide in the milk powder of example one are also tested, and the test results are shown in table 3 below.
The test results in Table 3 show that the average recovery rates of the additives at concentrations of 50. mu.g/mL, 200. mu.g/mL and 500. mu.g/mL exceed 95%, which meets the requirements.
TABLE 3 dicyandiamide addition concentration and recovery range in milk powder
Example two:
in this example, the type of sample used was a certain brand of defatted flour.
The adopted pipettor specification and numbering: 018583G (1mL)/R29762F (100 μ L).
The instrument name used: thermo liquid chromatography mass spectrometer.
The method for determining the residual amount of dicyandiamide in the dairy product mainly comprises the following steps:
s1: preparing dicyandiamide standard working solutions with different concentrations;
s2: performing ultrasonic extraction on residual dicyandiamide in a dairy product (defatted powder of a certain brand in the embodiment) by using a mixed solution of acetonitrile and water, and performing matrix dispersion solid-phase extraction and purification to prepare a to-be-detected liquid;
s3: performing mass spectrometry on the dicyandiamide standard working solution prepared in the step S1 by using a liquid chromatography tandem mass spectrometer, and drawing a standard curve;
s4: and (4) performing mass spectrometry on the liquid to be detected in the step S2 by using a liquid chromatography tandem mass spectrometer, and obtaining the content of the dicyandiamide in the dairy product through the standard curve of the step S3.
In this embodiment, the method for determining the residual amount of dicyandiamide in the dairy product of the present invention includes the preparation of a standard working solution of dicyandiamide. More specifically, the preparation of dicyandiamide standard working solution includes:
s11, preparing dicyandiamide internal standard stock solution: accurately weighing a proper amount of dicyandiamide internal standard (accurate to 0.1mg), and preparing a standard stock solution with the concentration of not less than 100 mug/mL (the standard stock solution can be stored for 1 year at 4 ℃) by using water;
s12, preparing dicyandiamide internal standard intermediate liquid: diluting the internal standard stock solution into a standard intermediate solution of 10 mug/mL;
s13, preparing dicyandiamide standard working solution: and diluting the standard intermediate solution of the dicyandiamide with water into standard working solution with the concentration of 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL and 50 ng/mL.
In one embodiment, preferably, the standard working fluid may also be formulated by: a dicyandiamide standard solution (concentration: 10. mu.g/mL) was taken at 100. mu.L, diluted with 900. mu.L of acetonitrile to give a standard solution at 1.0. mu.g/mL, 100. mu.L was taken, diluted with 900. mu.L of acetonitrile to give a standard solution at 100ng/mL, 20. mu.L, 50. mu.L, 80. mu.L, 100. mu.L, 200. mu.L, and 500. mu.L were taken, 20. mu.L of a dicyandiamide isotope (1.0. mu.g/mL) was added to each of the sample bottles, and the samples were diluted with 960. mu.L, 930. mu.L, 900. mu.L, 880. mu.L, 780. mu.L, and 480. mu.L of acetonitrile to give standard working solutions at concentrations of 0.002. mu.g/mL, 0.005. mu.g/mL, 0.008. mu.g/mL, 0.010. mu.g/mL, 0.020. mu. The standard curves of 0.002. mu.g/mL, 0.005. mu.g/mL, 0.008. mu.g/mL, 0.010. mu.g/mL, 0.020. mu.g/mL and 0.050. mu.g/mL were obtained by measurement with a Thermo liquid chromatography-mass spectrometer.
Preferably, the method for determining the residual amount of dicyandiamide in the dairy product further comprises the following steps of sample preparation and preservation: and (3) putting about 200g of a sample into a clean container as a sample, sealing and marking, and storing at normal temperature.
Further, the step S2 of the method for determining the residual amount of dicyandiamide in the dairy product of the present invention for extracting the residual dicyandiamide in the dairy product includes the following steps:
s21: weighing a sample, adding water, performing ultrasonic treatment, adding n-hexane saturated acetonitrile and acetonitrile saturated n-hexane, performing vortex mixing uniformly, oscillating, centrifuging, and extracting an acetonitrile layer.
Preferably, in this embodiment, the step S2 of extracting the residual dicyandiamide in the dairy product includes the following steps:
weighing 1g-5g of sample, adding 2mL-10mL of water, performing ultrasonic treatment for 5min-15min, adding 10mL-30mL of n-hexane saturated acetonitrile and 5mL-15mL of acetonitrile saturated n-hexane, performing vortex mixing uniformly, performing extraction for 10min-30min by oscillation, and centrifuging for 2min-8min at 10000r/min-18000 r/min; and extracting the acetonitrile layer.
In a preferred embodiment, the extraction of residual dicyandiamide in the dairy product in step S2 mainly comprises the following steps:
weighing about 2g of sample (accurate to 0.001g), adding 20 mu L of internal standard, adding 3mL of water, performing ultrasonic treatment for 10min, adding 17mL of n-hexane saturated acetonitrile, performing vortex mixing on 10mL of acetonitrile saturated n-hexane uniformly, performing oscillation extraction for 20min, and centrifuging for 3min at 15000 r/min; and extracting the acetonitrile layer.
In this embodiment, the purification of the acetonitrile layer in step S2 mainly includes the following steps:
and (4) adding the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with anhydrous magnesium sulfate, C18, PSA and GCB, uniformly mixing by vortex, oscillating, centrifuging, taking the supernatant into a small centrifuge tube, centrifuging again, and taking the supernatant to be tested.
In a preferred embodiment, the purification of the acetonitrile layer in step S2 comprises the steps of:
adding 1-5 mL of the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with 200-600 mg of anhydrous magnesium sulfate, 60-150 mg of C18, 80-150 mg of PSA and 30-100 mg of GCB, uniformly mixing by vortex, oscillating for 8-15 min, centrifuging for 1-10 min at 10000-18000 r/min, taking 0.8-2 mL of supernatant to a small centrifuge tube, centrifuging for 1-10 min at 10000-18000 r/min, taking the supernatant and bottling to be tested.
In a further preferred embodiment, the purification of the acetonitrile layer in step S2 comprises the steps of: weighing 400mg of anhydrous magnesium sulfate, 100mgC18 mg of 100mg of PSA and 50mg of GCB respectively, putting the anhydrous magnesium sulfate, 100mgC18 mg of PSA and 50mg of GCB into a 5mL centrifuge tube, adding 2.5mL of acetonitrile layer obtained by extraction into the 5mL centrifuge tube, uniformly mixing by vortex, oscillating for 10min, centrifuging for 3min at 12000r/min, adding 1.0mL of supernatant into a 2mL small centrifuge tube, centrifuging for 3min at 12000r/min, taking the supernatant, bottling, and measuring by a computer.
In this embodiment, preferably, the sample of defatted powder is extracted with acetonitrile and water saturated with n-hexane, the acetonitrile layer is purified with a purification tube containing magnesium sulfate, PSA, C18 and graphitized carbon, and the purified acetonitrile is centrifuged through a membrane and then loaded on a machine. Sample dilution factor: 20; volume fixing of the sample: 1 mL.
In this embodiment, the working conditions of the liquid chromatography-mass spectrometer include:
liquid chromatography column: cation exchange and C18 mixed column CR (1: 4) 2.0X 150mm X5 μm;
mobile phase: mobile phase C (20mmol/L ammonium acetate + 0.2% formic acid) to mobile phase B (acetonitrile) 90: 10;
flow rate: 0.3 mL/min;
sample introduction amount: 5 mu L of the solution;
column temperature: 35 ℃ is carried out.
In this example, unless otherwise specified, the mass spectrometry conditions listed in table 1 of example one and the measurement operating conditions for multi-reaction monitoring ion pairs, collision gas energy, cone-hole voltage, and residence time shown in table 2 were selected.
Further, the method for determining the residual amount of dicyandiamide in the dairy product comprises the following steps:
41) and (4) mass spectrometry. The method comprises the step of respectively measuring a sample to be measured and a standard working solution by using a liquid chromatography tandem mass spectrometer. And (4) drawing a standard curve by taking the concentration of the dicyandiamide in the standard working solution, the concentration of the internal standard and the corresponding peak area ratio as a vertical coordinate.
In one embodiment, the method for determining the residual amount of dicyandiamide in the dairy product further comprises the following steps:
42) and (5) blank testing. Selecting a negative sample, and performing the mass spectrometry step of 41) above.
In one embodiment, the method for determining the residual amount of dicyandiamide in the dairy product comprises the following steps:
43) the content of dicyandiamide in the sample is calculated according to the formula:
in the formula: x represents the content of dicyandiamide in the sample, mg/kg;
c represents the concentration of the component to be measured in the sample solution, ng/mL, as measured from the standard curve;
m represents the sample mass, g;
note: blank values need to be deducted from the calculation result.
Further, the related chromatograms of the second embodiment are shown in fig. 5 to 8. FIG. 5 shows a standard solution chromatogram of dicyandiamide, 0.01. mu.g/mL, of example two. The retention time is shown to be 1.47min, and the signal-to-noise ratio is more than 10. Has good resolution and sensitivity.
FIG. 6 shows a sample chromatogram of defatted flour of example two; in the chromatogram, dicyandiamide was not detected in the blank sample.
FIG. 7 shows a blank sample chromatogram of defatted flour; in the chromatogram, it is shown that dicyandiamide in the blank sample is not detected (see table 4 below) and there is no other interference.
FIG. 8 is a chromatogram of a blank sample of milk powder with a standard sample recovered; in the chromatogram, the retention time of dicyandiamide is shown to be 1.48min, the signal-to-noise ratio of quantitative ions is more than 10, the recovery rate is more than or equal to 90 percent (refer to the following table 5), the quality control requirement of a laboratory is met, and no other interference exists.
In addition, the detection results of dicyandiamide in the defatted powder sample obtained by the method for determining the residual dicyandiamide in the dairy product are shown in the following table 4:
TABLE 4 detection results of dicyandiamide in defatted powder samples
In Table 4, the concentration is measured in mg/kg. The lower limit of the dicyandiamide in the invention is 0.05 mg/kg. The results of the measurements were less than the lower limit of the measurement, and all were expressed as < 0.05 mg/kg. The test results in Table 4 show that the test results are satisfactory.
In addition, in this embodiment, the standard recovery rate of dicyandiamide in the defatted powder sample obtained by the method for determining the residual amount of dicyandiamide in the dairy product according to the present invention is shown in table 5:
TABLE 5 recovery of dicyandiamide from spent meal samples
The unit of constant volume in table 5 is mL; the concentration units were determined as mg/kg. Table 5 shows that the recovery rate of dicyandiamide in the degreasing powder sample is more than or equal to 90 percent.
As can be seen from tables 4 and 5 above: the residual amount of dicyandiamide in the degreased powder is detected by applying the method of the invention, and the detection result is undetected. In the practical detection of the skim milk powder, two added and recovered samples with concentration levels of 0.1mg/kg have good recovery rates, the requirement of experimental quality control can be met, and the scientific and accurate detection result is ensured. The method has good applicability in practical detection.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, the appended claims are intended to be construed to include preferred embodiments and all such changes and/or modifications as fall within the scope of the invention, and all such changes and/or modifications as are made to the embodiments of the present invention are intended to be covered by the scope of the invention.
Claims (10)
1. The method for measuring the residual amount of dicyandiamide in the dairy product is characterized by comprising the following steps of:
s1: preparing dicyandiamide standard working solutions with different concentrations;
s2: ultrasonically extracting residual dicyandiamide in the dairy product by using a mixed solution of acetonitrile and water, and performing matrix dispersion solid phase extraction and purification to prepare a liquid to be detected;
s3: performing mass spectrometry on the dicyandiamide standard working solution prepared in the step S1 by using a liquid chromatography tandem mass spectrometer, and drawing a standard curve;
s4: and (4) performing mass spectrometry on the liquid to be detected in the step S2 by using a liquid chromatography tandem mass spectrometer, and obtaining the content of the dicyandiamide in the dairy product through the standard curve of the step S3.
2. The method according to claim 1, wherein the step of preparing the dicyandiamide standard working solution in the step S1 includes the steps of:
s11: taking a proper amount of dicyandiamide internal standard substance, and preparing a standard stock solution with the concentration of not less than 100 mug/mL by using pure water;
s12: diluting the stock solution into a standard intermediate solution with 10 mug/mL by using a pure water solution;
s13: and diluting the intermediate solution with water to obtain standard working solution with various concentrations.
3. The method for determining dicyandiamide in claim 2, wherein the step of extracting dicyandiamide remaining in the dairy product in step S2 includes the steps of:
s21: weighing a sample, adding water, performing ultrasonic treatment, adding n-hexane saturated acetonitrile and acetonitrile saturated n-hexane, performing vortex mixing uniformly, oscillating, centrifuging, and extracting an acetonitrile layer.
4. The method for determining dicyandiamide in claim 3, wherein the step S2 of extracting residual dicyandiamide in the dairy product comprises the following steps:
weighing 1g-5g of sample, adding 2mL-10mL of water, performing ultrasonic treatment for 5min-15min, adding 10mL-30mL of n-hexane saturated acetonitrile and 5mL-15mL of acetonitrile saturated n-hexane, performing vortex mixing uniformly, performing extraction for 10min-30min by oscillation, and centrifuging for 2min-8min at 10000r/min-18000 r/min; and extracting the acetonitrile layer.
5. The method according to claim 4, wherein the purification of the acetonitrile layer in step S2 comprises the steps of:
and (4) adding the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with anhydrous magnesium sulfate, C18, PSA and GCB, uniformly mixing by vortex, oscillating, centrifuging, taking the supernatant into a small centrifuge tube, centrifuging again, and taking the supernatant to be tested.
6. The method according to claim 5, wherein the purification of the acetonitrile layer in step S2 comprises the steps of:
adding 1-5 mL of the acetonitrile layer extracted in the step S21 into a centrifuge tube filled with 200-600 mg of anhydrous magnesium sulfate, 60-150 mg of C18, 80-150 mg of PSA and 30-100 mg of GCB, uniformly mixing by vortex, oscillating for 8-15 min, centrifuging for 1-10 min at 10000-18000 r/min, taking 0.8-2 mL of supernatant to a small centrifuge tube, centrifuging for 1-10 min at 10000-18000 r/min, taking the supernatant and bottling to be tested.
7. The method according to claim 5 or 6, wherein the step S4 of obtaining the dicyandiamide content according to the standard curve of the step S3 includes:
s41: determining the liquid to be detected in the step S2 by adopting a liquid chromatography tandem mass spectrometer to obtain the peak area of the liquid to be detected;
s42: the concentration c of the component to be measured in the sample solution is obtained by checking a standard curve according to the peak area;
s43: according to the formula
Calculating the content of dicyandiamide in the sample, wherein c represents the concentration of the component to be detected in the sample solution, ng/mL, which is obtained by looking up a standard curve; m represents the sample mass, g; x represents the dicyandiamide content in the sample, mg/kg.
8. The assay according to claim 5 or 6, further comprising a blank test, wherein the determination of the blank test comprises:
and selecting a negative sample, and performing mass spectrometry by adopting a liquid chromatography tandem mass spectrometer according to the step of measuring the standard working solution to obtain a mass spectrogram of a blank test.
9. The assay according to any one of claims 1 to 8, wherein the chromatographic conditions employed in steps S3 and S4 comprise:
a1) a chromatographic column: waters BEH Amide, 1.7 μm × 2.1mm × 50 mm;
b1) temperature of the column: 30 ℃;
c1) mobile phase: (5mmol/L amine acetate + 0.1% formic acid)/acetonitrile (5/95);
d1) flow rate: 0.2 mL/min;
e1) sample introduction amount: 2.5 mu L;
or
a2) Liquid chromatography column: cation exchange and C18 mixed column CR (1: 4) 2.0X 150mm X5 μm;
b2) column temperature: 35 ℃;
c2) mobile phase: mobile phase C (20mmol/L ammonium acetate + 0.2% formic acid) to mobile phase B (acetonitrile) 90: 10;
d2) flow rate: 0.3 mL/min;
e2) sample introduction amount: 5 μ L.
10. The assay of any one of claims 1 to 7, wherein the mass spectrometry conditions employed in steps S3 and S4 comprise:
an ionization mode: ESI +;
taper hole airflow: nitrogen gas, 40L/h;
capillary voltage: 3.0 kV;
desolventizing the gas stream: nitrogen gas, 1000L/h;
source temperature: 150 ℃;
collision gas: argon gas;
the temperature of the desolvation: 500 ℃;
and (3) monitoring mode: multiple Reaction Monitoring (MRM); and/or
The multi-reaction monitoring ion pairs, collision gas energy, cone hole voltage and residence time adopted in the steps S3 and S4 comprise:
dicyandiamide compound, parent ion 84.8 and daughter ion 67.75 are adopted; the adopted taper hole voltage is 10V, and the collision energy is 13 eV;
dicyandiamide compound, parent ion 84.8 and daughter ion 45.8 are adopted; the adopted taper hole voltage is 13V, and the collision energy is 13 eV;
adopting dicyandiamide internal standard compound, mother ion 88.8 and daughter ion 70.75; the cone hole voltage is 25V, and the collision energy is 13 eV.
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