The content of the invention
Based on this, it is necessary to for the not high problem of the accuracy and reliability of traditional dried meat floss Product checking, there is provided
A kind of authentication method of dried meat floss product of poor quality.
The technical scheme that the present invention solves above-mentioned technical problem is as follows.
A kind of authentication method of dried meat floss product of poor quality, comprises the steps:
Step one:Add organic solvent to extract fat constituent therein in the sample of dried meat floss product, volatilize described organic
Solvent, collects solid phase;
Step 2:Acetonitrile solution is added in the solid phase obtained to step one, pigment composition therein is extracted, centrifugation is received
Collection liquid solution;
Step 3:Ammonium sulfate is added after the liquid solution concentration that step 2 is collected, is removed wherein with precipitating
Protein component, centrifugation, collect liquid solution;
Step 4:The liquid solution that step 3 is collected is blown to after net doing, polyamide solid phase extraction cartridge is proceeded to, to remove it
In natural colouring matter composition, collect liquid solution;
Step 5:The liquid solution collected in step 4 is blown to it is net dry, with water transfer it is fixed it is molten after cross high performance liquid chromatography
Instrument, using PDAD synthetic dyestuff content therein is detected, quantified by external standard method qualitative with retention time is obtained
Qualification result.
Wherein in one embodiment, in the step one, organic solvent used be initial boiling point be not less than 30 DEG C and
The end point of distillation is not higher than 60 DEG C of petroleum ether.
Wherein in one embodiment, in the step 2, the volumetric concentration of the acetonitrile solution is 70%;
Described to extract pigment composition therein be will to add the solution for having the acetonitrile solution with ultrasonically treated, so as to contain
There is pigment composition to be dissolved in the acetonitrile solution, then be centrifuged, and repeat to extract repeatedly until centrifugation with the acetonitrile solution
Extracting liquid colourless afterwards, merges extract and obtains liquid solution.
Wherein in one embodiment, in the step 3, the liquid solution concentration that step 2 is collected
Ammonium sulfate is added to include afterwards:After by liquid solution heating rotary evaporation concentration, the institute that mass concentration is 10% is added while hot
State ammonium sulfate.
Wherein in one embodiment, in the step 3, also include:The precipitation obtained using water washing centrifugation, then
Liquid solution is collected by centrifugation, and merges each centrifugate and obtain liquid solution.
It is described that the liquid solution that step 3 is collected is blown to into net doing in the step 4 wherein in one embodiment
It is that the liquid solution is blown to into net doing using nitrogen;
Described to proceed to polyamide solid phase extraction cartridge, to remove natural colouring matter composition therein, collecting liquid solution includes:Will
Be blown to net dry solution to proceed to after polyamide solid phase extraction cartridge, first using 60 DEG C of pH 4.0 solution washing described in polyamide
Solid-phase extraction column is repeatedly, then colourless to stripping liquid using absolute ethyl alcohol-ammoniacal liquor-solution washing, merges each stripping liquid and obtains
Liquid solution.
Wherein in one embodiment, the aqueous solution of the pH 4.0 is the water that ultra-pure water to pH 4.0 is adjusted with citric acid
Solution;
The volume ratio of absolute ethyl alcohol, ammoniacal liquor and water is 7 in the absolute ethyl alcohol-ammoniacal liquor-aqueous solution:2:1.
It is described to be blown to the liquid solution collected in step 4 only in the step 5 wherein in one embodiment
It is dry, included with the fixed molten rear high performance liquid chromatograph of crossing of water transfer:By the liquid solution collected in step 4 with second acid for adjusting pH extremely
7.0, blow steaming to net dry using nitrogen, with water transfer it is fixed it is molten after cross high performance liquid chromatograph.
Wherein in one embodiment, in the step 5, also include by water transfer it is fixed it is molten after the solution mistake that obtains
The step of 0.22 μm of membrane filtration, test liquid of the solution obtained after filtration as excessively described high performance liquid chromatograph.
Wherein in one embodiment, in the step 5, the condition of chromatogram is:
Chromatographic column:Size is the octadecylsilane chemically bonded silica post of 5mm*4.6mm*250mm;
Mobile phase:Volume ratio is 15:85 methyl alcohol and concentration for the ammonium acetate solution of 20mmol/L mixed liquor;
Flow velocity:1mL/min;
Column temperature:40℃;
PDAD wavelength used when detecting is 450nm.
Whether the authentication method of above-mentioned dried meat floss of poor quality differentiates dried meat floss by the method for colouring agent (pigment) in detection dried meat floss
It is adulterated, can simultaneously detect the synthetic dyestuffs such as sunset yellow, lemon yellow, famille rose, Wang Jinhuang in dried meat floss, by detecting dried meat floss in have
Whether no added pigment is adulterated to differentiate dried meat floss.The authentication method is readily soluble in water using pigment, in most of organic solvent
The minimum characteristic of solubility removes first fat constituent, makes protein coagulative precipitation using the method saltoutd and removes isolating protein
Interference, using the interference of solid phase extraction Polysaccharide removing and natural colouring matter, and concentrating sample solution.The authentication method solves king
The low problem of the golden yellow SPE rate of recovery, good separating effect, specificity is strong, and detection limit and quantitative limit are sufficiently low, the range of linearity
Well, it is reproducible, accurately and reliably, can be used for day-to-day supervision detection.
Specific embodiment
For the ease of understanding the present invention, the present invention is described more fully below in conjunction with accompanying drawing.But, the present invention
Can realize in many different forms, however it is not limited to embodiment described herein.On the contrary, providing these embodiments
Purpose is to make the understanding to the disclosure more thorough comprehensive.
Unless otherwise defined, all of technology used herein and scientific terminology and the technical field for belonging to the present invention
The implication that technical staff is generally understood that is identical.The term for being used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that of the invention in limiting.Term as used herein "and/or" includes one or more phases
The arbitrary and all of combination of the Listed Items of pass.
The authentication method of the dried meat floss product of poor quality of one embodiment, comprises the steps:
Step one:Add organic solvent to extract fat constituent therein in the sample of dried meat floss product, volatilize organic solvent,
Collect solid phase.
Organic solvent used is that initial boiling point is not less than 30 DEG C and the end point of distillation is not higher than 60 DEG C of petroleum ether.
Step 2:Acetonitrile solution is added in the solid phase obtained to step one, pigment composition therein is extracted, centrifugation is received
Collection liquid solution.
The volumetric concentration of acetonitrile solution is preferably 70%.
It is will to add the solution for having acetonitrile solution with ultrasonically treated specifically to extract pigment composition therein, such as ultrasonic
10min, so as to be dissolved in acetonitrile solution containing pigment composition, then is centrifuged, and such as 5min is centrifuged with 10000r/min, and uses acetonitrile
The aqueous solution repeats to extract repeatedly until the extracting liquid colourless after centrifugation, merges extract and obtain liquid solution.
Step 3:Ammonium sulfate is added after the liquid solution concentration that step 2 is collected, is removed wherein with precipitating
Protein component, centrifugation, collect liquid solution.
Specifically, liquid solution heating rotary evaporation concentration such as can be removed partial solvent in 60 DEG C of rotary evaporations, then
The ammonium sulfate that mass concentration is 10% is added while hot.
Preferably, step 3 also includes the precipitation obtained using water washing centrifugation, then liquid solution is collected by centrifugation, and merges
Each centrifugate obtains liquid solution.
Step 4:The liquid solution that step 3 is collected is blown to after net doing, polyamide solid phase extraction cartridge is proceeded to, to remove it
In natural colouring matter composition, collect liquid solution.
It is described to be blown to the liquid solution that step 3 is collected only dry to be to be blown to the liquid solution using nitrogen to do only, to remove
Remove unnecessary organic principle, concentrating sample.
Described to proceed to polyamide solid phase extraction cartridge, to remove natural colouring matter composition therein, collecting liquid solution includes:Will
It is blown to net dry solution to proceed to after polyamide solid phase extraction cartridge, first using the solution washing polyamide solid phase of 60 DEG C of pH 4.0
Extraction column is repeatedly, then colourless to stripping liquid using absolute ethyl alcohol-ammoniacal liquor-solution washing, merges each stripping liquid and obtains liquid phase
Solution.Preferably, the aqueous solution of pH 4.0 is the aqueous solution that ultra-pure water to pH 4.0 is adjusted with citric acid;Absolute ethyl alcohol-ammoniacal liquor-
The volume ratio of absolute ethyl alcohol, ammoniacal liquor and water is 7 in the aqueous solution:2:1.
Step 5:The liquid solution collected in step 4 is blown to it is net dry, with water transfer it is fixed it is molten after cross high performance liquid chromatography
Instrument, using PDAD synthetic dyestuff content therein is detected, quantified by external standard method qualitative with retention time is obtained
Qualification result.
It is described the liquid solution collected in step 4 is blown to it is net dry, with water transfer it is fixed it is molten after cross high performance liquid chromatograph bag
Include:By the liquid solution collected in step 4 second acid for adjusting pH to 7.0, steaming is blown to net dry, remove ammoniacal liquor and ethanol using nitrogen,
Molten rear high performance liquid chromatograph excessively calmly is shifted with water.
Preferably, in step 5, also include by water transfer it is fixed it is molten after the solution that obtains cross 0.22 μm of membrane filtration
Step, the solution obtained after filtration is used as the test liquid for crossing high performance liquid chromatograph.
The condition of chromatogram is preferably but not limited to:
Chromatographic column:Size is the octadecylsilane chemically bonded silica post of 5mm*4.6mm*250mm.
Mobile phase:Volume ratio is 15:85 methyl alcohol and concentration for the ammonium acetate solution of 20mmol/L mixed liquor.
Flow velocity:1mL/min.
Column temperature:40℃.
PDAD wavelength used when detecting is preferably but not limited to as 450nm.
It is below specific embodiment part.
1. instrument and reagent
1.1 high performance liquid chromatographs (carrying PDAD).
1.2 standard substance:Sunset yellow, lemon yellow, Wang Jinhuang, famille rose.
1.3 AR:Ammonium acetate, ammonium sulfate, petroleum ether (30~60 DEG C), ethanol, formic acid, acetic acid, citric acid.
1.4 chromatogram pure reagents:Methyl alcohol, acetonitrile.
1.5 polyamide solid phases extract pillar.
The water of 1.6pH=4, ultra-pure water is adjusted to pH=4 with citric acid.
1.7 methyl alcohol formic acid mixed liquors:Methyl alcohol:Formic acid=6:4 (volume ratios).
1.8 absolute ethyl alcohols-ammoniacal liquor-aqueous solution:Absolute ethyl alcohol:Ammoniacal liquor:Water=7:2:1 (volume ratio).
2. the preparation of standard liquid
Polychrom mixing storing solution:Weigh standard substance appropriate, with water dissolves and be diluted to concentration and be about 1mg/mL's
Storing solution.
Polychrom standard solution:Take the standard solution that storing solution is diluted with water to the μ g/mL of concentration about 50.
3. sample detection
3.1 take dried meat floss outturn sample about 10g, in putting 100mL erlenmeyer flasks, plus petroleum ether 50mL, stir and discard petroleum ether,
In triplicate, volatilize, add 70% acetonitrile solution ultrasonic extraction 10min, 10000r/min centrifugation 5min, extract proceed to from
Heart flask, repeats extraction process to extracting liquid colourless.
3.2 merge extract, in 60 DEG C of rotary evaporations to 5mL, the ammonium sulfate solutions of 1mL 10% are added while hot,
10000r/min is centrifuged 5min, and supernatant proceeds to new centrifuge tube, is precipitated with a little water washing, and centrifugation merges centrifugate.
Centrifugate is blown to net doing by 3.3 using nitrogen.
Above-mentioned solution is proceeded to polyamide solid phase extraction cartridge by 3.4, first using the solution washing 3 times of 60 DEG C of pH 4.0,
Then it is colourless to stripping liquid using absolute ethyl alcohol-ammoniacal liquor-solution washing.
3.5 merge stripping liquid, and using second acid for adjusting pH to 7.0, nitrogen blows steaming to net dry, and with water transfer 20mL, mistake are settled to
0.22 μm of filter membrane, as test liquid.
3.6 take standard liquid and each 10 μ L of sample solution inject high performance liquid chromatograph, by the way that retention time is qualitative, peak face
Product is quantitative.
Chromatographic condition:
Chromatographic column:Octadecylsilane chemically bonded silica post, 5*4.6*250mm
Mobile phase:Methyl alcohol:20mmol/L ammonium acetate solution=15:85 (volume ratios)
Flow velocity:1mL/min column temperatures:40℃
Detector:PDAD wavelength:450nm
3.6 result judgements, detect that any one of sunset yellow, lemon yellow, famille rose and Wang Jinhuang pigment can be sentenced
It is set to dried meat floss product of poor quality.
4. method validation
4.1 separating degree
The collection of illustrative plates of spiked levels three injection liquid phase high performance chromatograph is gone to be analyzed, separating degree result such as table 1 below.Standard liquid
Collection of illustrative plates is shown in Fig. 1, and sample solution collection of illustrative plates is shown in Fig. 2.
Table 1
Ingredient names |
Lemon yellow |
It is carmine |
Sunset yellow |
Wang Jinhuang |
Separating degree |
--- |
45 |
14 |
63 |
4.2 detection limits and quantitative limit
Take standard liquid to be tested, as detection limit, concentration when signal to noise ratio is 10 is fixed to concentration when with signal to noise ratio being 3
Amount limit, as a result such as table 2 below.
Table 2
Ingredient names |
Lemon yellow |
It is carmine |
Sunset yellow |
Wang Jinhuang |
Concentration (μ g/mL) |
2.311 |
2.186 |
0.981 |
1.219 |
Signal to noise ratio |
68.04 |
46.27 |
40.56 |
94.18 |
Detection limit (mg/kg) |
0.21 |
0.29 |
0.15 |
0.08 |
Quantitative limit (mg/kg) |
0.68 |
0.95 |
0.49 |
0.26 |
3.3 ranges of linearity, are shown in Table 3.
Table 3
The range of linearity of lemon yellow:2.3 μ g/mL~23 μ g/mL, linearly dependent coefficient:1.0000;
The carmine range of linearity:2.2 μ g/mL~22 μ g/mL, linearly dependent coefficient:1.0000;
The range of linearity of sunset yellow:0.98 μ g/mL~9.8 μ g/mL, linearly dependent coefficient:1.0000;
The range of linearity of Wang Jinhuang:1.2 μ g/mL~12 μ g/mL, linearly dependent coefficient:0.9997.
4.4 precision, are shown in Table 4.
Table 4
4.5 the degree of accuracy
Respectively weigh during three parts of sample 10g (being accurate to 0.001g) being well mixed are placed in small beaker, add according to following table and mark
Quasi- solution, according to 2.2.3 test liquid is prepared, and is tested, and calculates the rate of recovery, the results are shown in Table 5.
Table 5
Between 91.315~98.40%, the carmine rate of recovery is 90.48%~97.37% for the rate of recovery of lemon yellow
Between, the rate of recovery of sunset yellow between 90.29%~99.00%, the rate of recovery of Wang Jinhuang between 92.50~99.31%,
Illustration method is accurately and reliably.
4.6 sample applicabilities
In market, stochastic buying two batches dried meat floss cake and a collection of dried meat floss cake, carefully remove dried meat floss, and inspection is carried out in the method
Work is surveyed, wherein a collection of detecting with the addition of sunset yellow, as a result such as table 6 below.
Table 6
Illustration method is suitable for the detection of dried meat floss sample.
Conclusion:Method is separated well, and specificity is strong, and detection limit and quantitative limit are sufficiently low, and the range of linearity is good, repeatability
It is good, accurately and reliably, can be used for day-to-day supervision detection.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but and
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that for one of ordinary skill in the art comes
Say, without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be defined by claims.