CN107271576A - The method for determining dicyandiamide residual quantity in dairy products - Google Patents
The method for determining dicyandiamide residual quantity in dairy products Download PDFInfo
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- CN107271576A CN107271576A CN201610219215.1A CN201610219215A CN107271576A CN 107271576 A CN107271576 A CN 107271576A CN 201610219215 A CN201610219215 A CN 201610219215A CN 107271576 A CN107271576 A CN 107271576A
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- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 title claims abstract description 125
- 238000000034 method Methods 0.000 title claims abstract description 81
- 235000013365 dairy product Nutrition 0.000 title claims abstract description 78
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 78
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000008267 milk Substances 0.000 claims abstract description 25
- 235000013336 milk Nutrition 0.000 claims abstract description 25
- 210000004080 milk Anatomy 0.000 claims abstract description 25
- 239000006228 supernatant Substances 0.000 claims abstract description 22
- 239000000843 powder Substances 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 16
- 239000011259 mixed solution Substances 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 13
- 238000005119 centrifugation Methods 0.000 claims description 12
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 150000001408 amides Chemical class 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 238000001819 mass spectrum Methods 0.000 claims description 7
- 238000012544 monitoring process Methods 0.000 claims description 7
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 7
- 239000003643 water by type Substances 0.000 claims description 7
- 238000004811 liquid chromatography Methods 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 36
- 102000004169 proteins and genes Human genes 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 238000011084 recovery Methods 0.000 description 16
- 150000002500 ions Chemical class 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 6
- 239000007789 gas Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229920000877 Melamine resin Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229940046892 lead acetate Drugs 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 2
- 239000000618 nitrogen fertilizer Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 241000108664 Nitrobacteria Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N anhydrous guanidine Natural products NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000009332 manuring Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 235000019633 pungent taste Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dairy Products (AREA)
Abstract
The invention discloses the method for dicyandiamide in dairy products of extracting, determine the method for dicyandiamide residual quantity and the method for determining dicyandiamide residual quantity in milk powder in dairy products.Extracting the method for dicyandiamide in dairy products includes：Dairy products are mixed and the first stewing process with water, the first mixed liquor is obtained；First mixed liquor is mixed and the second stewing process with acetic acid, then the second obtained mixed liquor is subjected to the first centrifugal treating, supernatant is collected；And mixed the supernatant and the second centrifugal treating with acetonitrile, obtain extract solution.Thus, the method for dicyandiamide can efficiently extract out dicyandiamide in extraction dairy products of the invention, and determine dicyandiamide residual quantity exactly according to the method for dicyandiamide residual quantity in measure dairy products of the invention.
Description
Technical Field
The invention relates to the field of food. In particular, the invention relates to a method for determining the residual amount of dicyandiamide in a dairy product. More particularly, the invention relates to a method for extracting dicyandiamide in a dairy product, a method for determining residual dicyandiamide in the dairy product and a method for determining residual dicyandiamide in milk powder.
Background
Dicyandiamide (dicyandiamide), a dimer of cyanamide, is also a cyano derivative of guanidine. Has a chemical formula of C2H4N4White crystalline powder. Dicyandiamide is a component of a composite nitrogenous fertilizer, can control the activity of nitrobacteria, adjust the conversion speed of a nitrogen fertilizer in soil, reduce the loss of nitrogen and improve the use efficiency of the fertilizer.
There are two routes for dicyandiamide to enter dairy products: the first possible approach is the active incorporation into dairy products by rogue merchants, like melamine, to counterfeit protein content. The dairy product not only lacks due nutritive value, but also causes the infant to develop badly, resulting in a series of health problems, so the dairy product is toxic milk powder which is extremely painful for people. The second possible route is the food chain route from chemical manuring to pasture and then to dairy cows, milk and milk powder. After the melamine event in china, dicyandiamide is listed as "milk necessity" by FDA.
However, the existing method for determining the residual amount of dicyandiamide in dairy products still needs to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention aims to provide a method for extracting dicyandiamide in a dairy product, a method for measuring the residual amount of dicyandiamide in the dairy product and a method for measuring the residual amount of dicyandiamide in milk powder. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide, and the residual dicyandiamide amount in the dairy product can be accurately measured according to the method for measuring the residual dicyandiamide amount in the dairy product.
It should be noted that the present invention has been completed based on the following findings of the inventors:
in the prior art, when acetonitrile is directly used for extracting dicyandiamide in a dairy product, the dicyandiamide can be coagulated together with protein while the acetonitrile precipitates the protein, so that the recovery rate is low, and the detection result is unstable.
In view of the above, the inventors have found through a large number of experiments that before acetonitrile is used to extract a dairy product, the dairy product dissolved in water is mixed with acetic acid to precipitate protein, and dicyandiamide does not precipitate together with the protein, so that acetonitrile can be ensured to effectively extract dicyandiamide, and then liquid chromatography-mass spectrometry is used to detect an extracting solution. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide, and the residual dicyandiamide amount in the dairy product can be accurately measured according to the method for measuring the residual dicyandiamide amount in the dairy product.
To this end, in a first aspect of the invention, the invention proposes a method for extracting dicyandiamide in a dairy product. According to an embodiment of the invention, the method comprises: mixing a dairy product with water and performing first standing treatment to obtain a first mixed solution; mixing the first mixed solution with acetic acid, performing second standing treatment, performing first centrifugal treatment on the obtained second mixed solution, and collecting supernatant; and mixing the supernatant with acetonitrile and carrying out second centrifugation treatment to obtain an extracting solution. The inventor finds that the first mixed solution is treated by acetic acid in advance, so that protein can be precipitated, and dicyandiamide exists in the supernatant, so that acetonitrile can be ensured to extract dicyandiamide from the supernatant, and protein interference is avoided. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
According to an embodiment of the invention, the concentration of acetic acid is 50% by volume. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
According to the embodiment of the invention, the mass ratio of the dairy product to the water is 1: 5-8. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
According to the embodiment of the invention, the mass ratio of the dairy product to the acetic acid is 6-8: 1. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
According to the embodiment of the invention, the time of the first standing treatment and the time of the second standing treatment are respectively 5-30 minutes independently. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
According to an embodiment of the invention, the time and the rotation speed of the first centrifugation and the second centrifugation are 5min to 10min and 10000rpm to 12000rpm, respectively and independently. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
According to an embodiment of the invention, the dairy product is milk powder. Therefore, the method for extracting dicyandiamide in the dairy product can fully extract dicyandiamide.
In a second aspect of the invention, the invention provides a method for determining the residual amount of dicyandiamide in a dairy product. According to an embodiment of the invention, the method comprises: extracting dicyandiamide from the dairy product according to the method for extracting dicyandiamide from the dairy product to obtain an extracting solution; and detecting the extracting solution by using a liquid chromatography-mass spectrometry method, and determining the residual amount of the dicyandiamide in the dairy product based on the detection result. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product according to the embodiment of the invention has high accuracy and strong stability.
According to an embodiment of the invention, the extract is subjected to a filtration process before the extract is detected by liquid chromatography-mass spectrometry, according to a preferred embodiment of the invention, the filtration process is performed with a 0.22 μm filter. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product according to the embodiment of the invention has high accuracy and strong stability.
According to the embodiment of the invention, the detection conditions of the liquid chromatography-mass spectrometry are as follows: liquid chromatography detection conditions: a chromatographic column: waters acquisition UPLC Amide; column temperature: 35 ℃; sample introduction volume: 5 mu L of the solution; gradient elution: the mobile phase at 0-1.5 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; the mobile phase at 1.5-3.5 min comprises: 50% by volume of mobile phase a and 50% by volume of mobile phase B; the mobile phase at 3.5-4.0 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; flow rate: 0.4ml/min, mass spectrometric detection conditions: an ionization mode: ESI (+); the ionization voltage is 3.0 KV; source temperature: 120 ℃; temperature of the desolventizing gas: 400 ℃; flow rate of desolventizing agent: 500L/hr; the collection mode is as follows: monitoring multiple reactions; taper hole voltage: 20V, and (3); and (3) quantitative ion pair: 85>68, collision energy: 22 eV; and (3) quantitative ion pair: 85>43, collision energy: 22 eV. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product according to the embodiment of the invention has high accuracy and strong stability.
In a third aspect of the invention, the invention provides a method for determining the residual amount of dicyandiamide in milk powder. According to an embodiment of the invention, the method comprises: (1) vortex oscillating 0.15g of sample and 0.85ml of water until the sample is completely dissolved, and standing for 5 min; (2) adding 20 mu l of 50% acetic acid into the mixed solution obtained in the step (1), carrying out vortex oscillation, standing for 5min, and then centrifuging for 5min at the rotating speed of 12000 rpm; (3) sucking 100 μ l of the supernatant obtained in step (2) and 900 μ l of acetonitrile, mixing by vortex oscillation, centrifuging at 12000rpm for 5min, and filtering the obtained supernatant with 0.22 μm filter to obtain a solution to be detected; (4) and (3) determining the liquid to be detected by using a liquid chromatography-tandem mass spectrum to obtain the peak area of the liquid to be detected, wherein the liquid chromatography detection conditions are as follows: a chromatographic column: waters acquisition UPLC Amide; column temperature: 35 ℃; sample introduction volume: 5 mu L of the solution; gradient elution: the mobile phase at 0-1.5 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; the mobile phase at 1.5-3.5 min comprises: 50% by volume of mobile phase a and 50% by volume of mobile phase B; the mobile phase at 3.5-4.0 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; flow rate: 0.4ml/min, mass spectrometric detection conditions: an ionization mode: ESI (+); the ionization voltage is 3.0 KV; source temperature: 120 ℃; temperature of the desolventizing gas: 400 ℃; flow rate of desolventizing agent: 500L/hr; the collection mode is as follows: monitoring multiple reactions; taper hole voltage: 20V, and (3); and (3) quantitative ion pair: 85>68, collision energy: 22 eV; and (3) quantitative ion pair: 85>43, collision energy: 22 eV; (5) substituting the peak area of the solution to be detected obtained in the step (4) into a formula, and calculating to obtain the residual amount of dicyandiamide in the sample, wherein the calculation formula is as follows: x ═ c × V × f × 1000/(m × 1000), X: residual dicyandiamide in the sample (μ g/kg); c: measuring the concentration (ng/mL) of dicyandiamide in the solution; v: sample volume to volume (mL); m: sample mass (g); f: a dilution factor. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product according to the embodiment of the invention has high accuracy and strong stability.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a standard graph according to one embodiment of the invention; and
FIG. 2 shows a chromatogram according to an embodiment of the invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The invention provides a method for extracting dicyandiamide in a dairy product, a method for measuring the residual amount of dicyandiamide in the dairy product and a method for measuring the residual amount of dicyandiamide in milk powder, which are respectively described in detail below.
Method for extracting dicyandiamide in dairy product
In a first aspect of the invention, the invention provides a method for extracting dicyandiamide in a dairy product. According to an embodiment of the invention, the method comprises: mixing a dairy product with water and performing first standing treatment to obtain a first mixed solution; mixing the first mixed solution with acetic acid, performing second standing treatment, performing first centrifugal treatment on the obtained second mixed solution, and collecting supernatant; and mixing the supernatant with acetonitrile and carrying out second centrifugation treatment to obtain an extracting solution.
In the prior art, the first mixed solution is directly mixed with acetonitrile to extract dicyandiamide. However, when acetonitrile is added, the protein is instantaneously coagulated into a mass, and dicyandiamide is coagulated together with the protein and is not easily dispersed in the supernatant, so that the recovery rate is low, and the detection result is unstable.
Therefore, the inventors have found through a large number of experiments that proteins can be precipitated by treating the first mixed solution with acetic acid before extracting acetonitrile, dicyandiamide is dispersed in a supernatant, and dicyandiamide in the supernatant is extracted with acetonitrile to obtain an extract solution, thereby avoiding interference of proteins. In addition, due to the volatility of acetic acid and acetonitrile, the subsequent liquid chromatography-mass spectrometry detection is facilitated. Acetic acid was selected by the inventors among numerous agents that precipitate proteins, but the other agents did not work well. For example, when the protein is precipitated by using lead acetate, since the subsequent mass spectrometry detection requires that the solvent in the sample solution is as volatile as possible, otherwise, the accuracy of the detection result is affected, and the service life of the mass spectrometry is also affected. Lead acetate is a non-volatile salt substance, and is not suitable for use. When the protein is precipitated by using the triple-filtration acetic acid, the acidity of the triple-filtration acetic acid is strong, so that the corrosion of an instrument is easily caused.
According to an embodiment of the invention, the concentration of acetic acid is 50% by volume. The inventors have optimized the above optimum acetic acid concentration through a number of experiments. Too high a concentration of acetic acid not only wastes reagents but also adversely affects experimental work due to the strong pungent taste of acetic acid.
According to the embodiment of the invention, the mass ratio of the dairy product to the water is 1: 5-8. According to another embodiment of the invention, the mass ratio of the dairy product to the acetic acid is 6-8: 1. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product can accurately determine the residual amount of dicyandiamide in the dairy product, and the result has strong stability.
It is to be noted that, according to the embodiment of the present invention, for liquid solvents such as water and acetic acid, 1mL is shown as 1g for convenience of calculation.
According to the embodiment of the invention, the time of the first standing treatment and the time of the second standing treatment are respectively 5-30 minutes independently. The inventors optimized the optimal first and second settling treatment times through a number of experiments. The purpose of the first standing treatment is to dissolve the sample sufficiently. Too short time and poor dissolution effect. The second standing treatment aims to ensure that the protein is fully denatured and precipitated under the action of acetic acid, if the standing time is too short, part of the protein is not completely precipitated, and the residual protein interferes subsequent operation, so that the accuracy of a detection result is influenced.
According to an embodiment of the invention, the time and the rotation speed of the first centrifugation and the second centrifugation are 5min to 10min and 10000rpm to 12000rpm, respectively and independently. The first centrifugation is intended to remove proteins, and if the centrifugation time is too short, the proteins cannot be completely removed, which may affect the subsequent detection. The purpose of the second centrifugation is to remove impurities insoluble in acetonitrile, so that dicyandiamide can be extracted with less impurities in the extract.
It should be noted that the invention does not strictly limit the kind of the dairy product, and according to the specific embodiment of the invention, the dairy product is milk powder.
Method for determining residual amount of dicyandiamide in dairy product
In a second aspect of the invention, the invention provides a method for determining the residual amount of dicyandiamide in a dairy product. According to an embodiment of the invention, the method comprises: extracting dicyandiamide from the dairy product according to the method for extracting dicyandiamide from the dairy product to obtain an extracting solution; and detecting the extracting solution by using a liquid chromatography-mass spectrometry method, and determining the residual amount of the dicyandiamide in the dairy product based on the detection result. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product can accurately determine the residual amount of dicyandiamide in the dairy product, and the result has strong stability.
According to an embodiment of the present invention, the extract is subjected to a filtration treatment before the detection of the liquid to be detected by liquid chromatography-mass spectrometry, and according to a preferred embodiment of the present invention, the filtration treatment is performed using a 0.22 μm filter. The filtering treatment is carried out to remove impurities with larger grain sizes, so as to prevent the impurities from influencing the detection result and avoid the damage to the instrument.
According to the embodiment of the present invention, the detection conditions of the liquid chromatography-mass spectrometry are as follows: liquid chromatography detection conditions: a chromatographic column: waters acquisition UPLC Amide; column temperature: 35 ℃; sample introduction volume: 5 mu L of the solution; gradient elution: the mobile phase at 0-1.5 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; the mobile phase at 1.5-3.5 min comprises: 50% by volume of mobile phase a and 50% by volume of mobile phase B; the mobile phase at 3.5-4.0 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; flow rate: 0.4ml/min, mass spectrometric detection conditions: an ionization mode: ESI (+); the ionization voltage is 3.0 KV; source temperature: 120 ℃; temperature of the desolventizing gas: 400 ℃; flow rate of desolventizing agent: 500L/hr; the collection mode is as follows: monitoring multiple reactions; taper hole voltage: 20V, and (3); and (3) quantitative ion pair: 85>68, collision energy: 22 eV; and (3) quantitative ion pair: 85>43, collision energy: 22 eV. Under the optimal detection condition, the dicyandiamide chromatographic peak is obvious, and no miscellaneous peak interference exists around the chromatographic peak, so that the residual dicyandiamide in the dairy product can be more accurately determined.
It will be understood by those skilled in the art that the features and advantages described above for the method for extracting dicyandiamide in dairy products are also applicable to the method for determining residual dicyandiamide content in dairy products, and will not be described in detail herein.
Method for measuring dicyandiamide residual quantity in milk powder
In a third aspect of the invention, the invention provides a method for determining the residual amount of dicyandiamide in milk powder. According to an embodiment of the invention, the method comprises: (1) vortex oscillating 0.15g of sample and 0.85ml of water until the sample is completely dissolved, and standing for 5 min; (2) adding 20 mu l of 50% acetic acid into the mixed solution obtained in the step (1), carrying out vortex oscillation, standing for 5min, and then centrifuging for 5min at the rotating speed of 12000 rpm; (3) sucking 100 μ l of the supernatant obtained in step (2) and 900 μ l of acetonitrile, mixing by vortex oscillation, centrifuging at 12000rpm for 5min, and filtering the obtained supernatant with 0.22 μm filter to obtain a solution to be detected; (4) measuring the liquid to be detected by using a liquid chromatogram-tandem mass spectrum to obtain the peak area of the liquid to be detected, wherein the detection conditions of the liquid chromatogram are as follows: a chromatographic column: waters acquisition UPLC Amide; column temperature: 35 ℃; sample introduction volume: 5 mu L of the solution; gradient elution: the mobile phase at 0-1.5 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; the mobile phase at 1.5-3.5 min comprises: 50% by volume of mobile phase a and 50% by volume of mobile phase B; the mobile phase at 3.5-4.0 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; flow rate: 0.4ml/min, mass spectrometric detection conditions: an ionization mode: ESI (+); the ionization voltage is 3.0 KV; source temperature: 120 ℃; temperature of the desolventizing gas: 400 ℃; flow rate of desolventizing agent: 500L/hr; the collection mode is as follows: monitoring multiple reactions; taper hole voltage: 20V, and (3); and (3) quantitative ion pair: 85>68, collision energy: 22 eV; and (3) quantitative ion pair: 85>43, collision energy: 22 eV; (5) substituting the peak area of the solution to be detected obtained in the step (4) into a formula, and calculating to obtain the residual amount of dicyandiamide in the sample, wherein the calculation formula is as follows: x ═ cxv × f × 1000/(m × 1000), X: residual dicyandiamide amount in the sample (μ g/kg); c, measuring the concentration (ng/mL) of dicyandiamide in the solution; v: sample volume to volume (mL); m: sample mass (g); f: a dilution factor. Therefore, the method for determining the residual amount of dicyandiamide in the dairy product according to the embodiment of the invention has high accuracy and strong stability.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, the method for determining the residual amount of dicyandiamide in milk powder was as follows:
(1) accurately weighing 0.15g of sample into a 2ml centrifuge tube, adding 0.85ml of ultrapure water, whirling to completely dissolve the sample, and standing for 5 min.
(2) Adding 20 mu l of 50% acetic acid into the mixed solution obtained in the step (1), swirling and standing for 5min, and centrifuging for 5min at the rotating speed of 12000 rpm.
(3) And (3) sucking 100 mu l of the supernatant obtained in the step (2) into another 2ml centrifuge tube, adding 900 mu l of acetonitrile, swirling, centrifuging at 12000rpm for 5min, collecting the supernatant, and filtering the supernatant through a 0.22 mu m filter to obtain a liquid to be detected.
(4) And (4) measuring the liquid to be measured by using a liquid chromatography-tandem mass spectrum to obtain the peak area of the liquid to be measured.
Wherein,
liquid chromatography detection conditions:
a chromatographic column: waters acquisition UPLC Amide;
column temperature: 35 ℃;
sample introduction volume: 5 mu L of the solution;
gradient elution: 0-1.5 min, 90 volume percent of mobile phase A and 10 volume percent of mobile phase B; 1.5-3.5 min, 50 volume percent of mobile phase A and 50 volume percent of mobile phase B; 3.5-4.0 min, 90 volume percent of mobile phase A and 10 volume percent of mobile phase B;
flow rate: 0.4ml/min of the mixture is added,
mass spectrum detection conditions:
an ionization mode: ESI (+);
the ionization voltage is 3.0 KV;
source temperature: 120 ℃;
temperature of the desolventizing gas: 400 ℃;
flow rate of desolventizing agent: 500L/hr;
the collection mode is as follows: monitoring multiple reactions;
taper hole voltage: 20V, and (3);
and (3) quantitative ion pair: 85>68, collision energy: 22 eV;
and (3) quantitative ion pair: 85>43, collision energy: 22 eV.
(5) Preparing dicyandiamide standard working solutions with six concentration gradients of 0ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL by using a blank matrix solution respectively, detecting according to the instrument conditions of the step (4), and obtaining a dicyandiamide standard curve (shown in figure 1) by using the peak area Y of the quantitative ions of the object to be detected to the mass concentration X (mu g/L), wherein the correlation coefficient is greater than 0.99, the linear relation is good, and the standard curve of an external standard method can be used for sample correction.
(6) Substituting the peak area of the solution to be detected obtained in the step (4) into a formula, and calculating to obtain the residual amount of the dicyandiamide in the sample.
Wherein, the calculation formula is:
x is the residual amount of dicyandiamide in the sample (mu g/kg);
c, measuring the concentration (ng/mL) of dicyandiamide in the solution;
v: sample volume to volume (mL);
m: sample mass (g);
f: a dilution factor.
Example 2
In this example, the recovery was determined according to the following procedure:
(1) milk without dicyandiamide was mixed with standards having dicyandiamide contents of 50. mu.g/kg, 100. mu.g/kg and 250. mu.g/kg, respectively, to obtain a spiked sample 1, a spiked sample 2 and a spiked sample 3.
(2) The residual amount of dicyandiamide in the different spiked samples was measured according to the method of example 1, and the recovery rate was calculated according to the following formula.
Specifically, the calculation formula is: the recovery (%) — actual measurement value of residual dicyandiamide amount in the spiked sample/theoretical value of residual dicyandiamide amount in the spiked sample.
The results of the recovery rate and the precision of dicyandiamide in milk are shown in Table 2. The recovery rate is within the range of 80-110%, and the recovery rate meets the requirement, which shows that the method has high accuracy. The relative standard deviation is in the range of 4.2-6.1%, which indicates that the precision is higher. FIG. 2 shows the chromatogram obtained for the spiked sample 1. As can be seen from the figure, no obvious interference of miscellaneous peaks exists around the dicyandiamide chromatographic peak, thereby ensuring the accuracy of the detection result.
Example 3
In this example, the recovery was determined according to the following procedure:
(1) mixing the milk powder without dicyandiamide with the standard substances with dicyandiamide contents of 400 mug/kg, 800 mug/kg and 2000 mug/kg respectively to obtain a standard sample 4, a standard sample 5 and a standard sample 6.
(2) The residual amount of dicyandiamide in the different spiked samples was measured according to the method of example 1, and the recovery rate was calculated according to the following formula.
Specifically, the calculation formula is: the recovery (%) — actual measurement value of residual dicyandiamide amount in the spiked sample/theoretical value of residual dicyandiamide amount in the spiked sample.
Table 3 shows the results of the dicyandiamide recovery and the precision measurement in the milk powder. The recovery rate is within the range of 80-110%, and the recovery rate meets the requirement, which shows that the method has high accuracy. The relative standard deviation is in the range of 4-6%, which shows that the precision is higher.
TABLE 2 detection of dicyandiamide recovery and precision in milk
TABLE 3 detection of dicyandiamide recovery and precision in milk powder
Comparative example 1
In this comparative example, the residual amount of dicyandiamide in the milk powder was measured in the same manner as in example 1 except that,
and (4) directly carrying out the operation of the step (3), namely directly adding 900 mu l of acetonitrile into 0.1 g of milk powder, then swirling, and carrying out subsequent operation.
Comparative example 2
In this comparative example, the residual amount of dicyandiamide in the milk powder was measured in the same manner as in example 1 except that,
step (2) is not included.
The results of the tests of comparative examples 1 and 2 are shown in table 4, and it can be seen that the methods of comparative examples 1 and 2 are low in accuracy and precision.
TABLE 4 comparison of dicyandiamide measurements from different extraction methods
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A method for measuring the residual amount of dicyandiamide in milk powder is characterized by comprising the following steps:
(1) vortex oscillating 0.15g of sample and 0.85ml of water until the sample is completely dissolved, and standing for 5 min;
(2) adding 20 mu l of 50% acetic acid into the mixed solution obtained in the step (1), carrying out vortex oscillation, standing for 5min, and then centrifuging for 5min at the rotating speed of 12000 rpm;
(3) sucking 100 μ l of the supernatant obtained in step (2) and 900 μ l of acetonitrile, mixing by vortex oscillation, centrifuging at 12000rpm for 5min, and filtering the obtained supernatant with 0.22 μm filter to obtain a solution to be detected;
(4) measuring the liquid to be measured by using a liquid chromatogram-tandem mass spectrum to obtain the peak area of the liquid to be measured,
wherein,
liquid chromatography detection conditions:
a chromatographic column: waters acquisition UPLC Amide;
column temperature: 35 ℃;
sample introduction volume: 5 mu L of the solution;
gradient elution: the mobile phase at 0-1.5 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; the mobile phase at 1.5-3.5 min comprises: 50% by volume of mobile phase a and 50% by volume of mobile phase B; the mobile phase at 3.5-4.0 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B;
flow rate: 0.4ml/min of the mixture is added,
mass spectrum detection conditions:
an ionization mode: ESI (+);
ionization voltage: 3.0 KV;
source temperature: 120 ℃;
temperature of the desolventizing gas: 400 ℃;
flow rate of desolventizing agent: 500L/hr;
the collection mode is as follows: monitoring multiple reactions;
taper hole voltage: 20V, and (3);
and (3) quantitative ion pair: 85>68, collision energy: 22 eV;
and (3) quantitative ion pair: 85>43, collision energy: 22 eV;
(5) substituting the peak area of the solution to be detected obtained in the step (4) into a formula, and calculating to obtain the residual amount of dicyandiamide in the sample, wherein the calculation formula is as follows: x is cxv × f × 1000/(m × 1000),
x: residual dicyandiamide in the sample (μ g/kg);
c: measuring the concentration (ng/mL) of dicyandiamide in the solution;
v: sample volume to volume (mL);
m: sample mass (g);
f: a dilution factor.
2. A method for extracting dicyandiamide in a dairy product is characterized by comprising the following steps:
mixing a dairy product with water and performing first standing treatment to obtain a first mixed solution;
mixing the first mixed solution with acetic acid, performing second standing treatment, performing first centrifugal treatment on the obtained second mixed solution, and collecting supernatant; and
and mixing the supernatant with acetonitrile, and carrying out second centrifugation treatment to obtain an extracting solution.
3. The method of claim 2,
the concentration of the acetic acid was 50% by volume,
optionally, the mass ratio of the dairy product to the water is 1: 5-8,
optionally, the mass ratio of the dairy product to the acetic acid is 6-8: 1.
4. The method according to claim 2, wherein the first standing treatment and the second standing treatment are each independently 5 to 30 minutes.
5. The method according to claim 2, wherein the first centrifugation and the second centrifugation are performed independently at a time and a rotation speed of 5min to 10min and 10000rpm to 12000rpm, respectively.
6. The method of claim 2, wherein the dairy product is a milk powder.
7. A method for measuring the residual quantity of dicyandiamide in a dairy product is characterized by comprising the following steps:
extracting dicyandiamide from a dairy product by the method of any one of claims 2 to 6 to obtain an extract; and
and detecting the extracting solution by using a liquid chromatography-mass spectrometry method, and determining the residual amount of the dicyandiamide in the dairy product based on the detection result.
8. The method according to claim 7, wherein the extract is subjected to a filtration treatment before the extract is detected by liquid chromatography-mass spectrometry,
preferably, the filtration treatment is performed using a 0.22 μm filter.
9. The method according to claim 7, wherein the detection conditions of the liquid chromatography-mass spectrometry are as follows:
liquid chromatography detection conditions:
a chromatographic column: waters acquisition UPLC Amide;
column temperature: 35 ℃;
sample introduction volume: 5 mu L of the solution;
gradient elution: the mobile phase at 0-1.5 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B; the mobile phase at 1.5-3.5 min comprises: 50% by volume of mobile phase a and 50% by volume of mobile phase B; the mobile phase at 3.5-4.0 min comprises: 90% by volume of mobile phase a and 10% by volume of mobile phase B;
flow rate: 0.4ml/min of the mixture is added,
mass spectrum detection conditions:
an ionization mode: ESI (+);
ionization voltage: 3.0 KV;
source temperature: 120 ℃;
temperature of the desolventizing gas: 400 ℃;
flow rate of desolventizing agent: 500L/hr;
the collection mode is as follows: monitoring multiple reactions;
taper hole voltage: 20V, and (3);
and (3) quantitative ion pair: 85>68, collision energy: 22 eV;
and (3) quantitative ion pair: 85>43, collision energy: 22 eV.
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|---|---|---|---|---|
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4918024A (en) * | 1987-11-13 | 1990-04-17 | Ulrich Rust | Method for determination of dicyanodiamide in plants or parts of plants |
| CN103217498A (en) * | 2013-04-23 | 2013-07-24 | 北京出入境检验检疫局检验检疫技术中心 | Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method |
| CN103344726A (en) * | 2013-06-14 | 2013-10-09 | 中华人民共和国温州出入境检验检疫局 | Method for extraction of dicyandiamide component in dairy product |
| CN105021737A (en) * | 2015-08-10 | 2015-11-04 | 云南省食品药品检验所 | Method for simultaneously detecting content of dicyandiamide and melamine in dairy products |
-
2016
- 2016-04-08 CN CN201610219215.1A patent/CN107271576A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4918024A (en) * | 1987-11-13 | 1990-04-17 | Ulrich Rust | Method for determination of dicyanodiamide in plants or parts of plants |
| CN103217498A (en) * | 2013-04-23 | 2013-07-24 | 北京出入境检验检疫局检验检疫技术中心 | Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method |
| CN103344726A (en) * | 2013-06-14 | 2013-10-09 | 中华人民共和国温州出入境检验检疫局 | Method for extraction of dicyandiamide component in dairy product |
| CN105021737A (en) * | 2015-08-10 | 2015-11-04 | 云南省食品药品检验所 | Method for simultaneously detecting content of dicyandiamide and melamine in dairy products |
Non-Patent Citations (3)
| Title |
|---|
| 刘红河等: "高效液相色谱-电喷雾串联质谱法测定奶及奶制品中双氰胺和三聚氰胺", 《卫生研究》 * |
| 张婷婷等: "反相高效液相色谱-串联质谱法测定母乳及奶粉中核苷酸的含量", 《分析化学》 * |
| 洪灯等: "高效液相色谱法检测塑料食品接触材料中的双氰胺", 《化学通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112505165A (en) * | 2020-07-10 | 2021-03-16 | 中检科（北京）测试技术有限公司 | Method for measuring residual quantity of dicyandiamide in dairy product |
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