CN109655567A - A kind of method of acid stain in detection animal meat products - Google Patents
A kind of method of acid stain in detection animal meat products Download PDFInfo
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- CN109655567A CN109655567A CN201811590217.7A CN201811590217A CN109655567A CN 109655567 A CN109655567 A CN 109655567A CN 201811590217 A CN201811590217 A CN 201811590217A CN 109655567 A CN109655567 A CN 109655567A
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a kind of methods of acid stain in detection animal meat products, this method carries out the extraction of sample to be tested using extracting solution, and by the supernatant obtained after extraction through WAX Solid Phase Extraction column purification, to complete sample pre-treatments, then ultra performance liquid chromatography-series connection quadrupole rod linear ion trap mass spectrometer is applied, establish 10 kinds of acid stain screenings simultaneously in animal meat products, the detection method of confirmation, the detection method has easy to operate, flux is efficient, it is qualitative accurate, high sensitivity, the features such as environmental-friendly, screening suitable for acid stains a variety of in meat products, confirmation, it can greatly improve detection efficiency, avoid the detection of false positive sample.
Description
Technical field
The present invention disclose be related to colorant detection technical field more particularly to a kind of detection animal meat products in acidity
The method of toner.
Background technique
Food colour can not only cause people to generate appetite, and be also an important organoleptic indicator.For example, consumer
When carrying out meat products purchase, it is that observation is yellowish pink first, using the yellowish pink judgment basis as freshness, and decides whether to purchase
It buys.Therefore, some meat products production firms enhance the desire to buy and appetite of consumer, lead to make the lovely luster of meat products
Colorant can often be added in meat products, and artificial close is added to cover the stale of raw meat for some illegal production firms
At colorant or even illegal addition industrial dye.
Artificial synthesized colorant lovely luster, strong coloring force, soluble, easy toning, chemical property stabilization, production cost
It is low, by processing and bright-coloured tempting color can be still kept after a long time placement, thus be widely applied by meat products manufacturer.But
Artificial synthesized colorant has certain toxicity, there is harm to the health of human body, so the addition to synthetic coloring matter is necessary
It is controlled.
Currently, the method for carrying out colorant detection mainly has liquid chromatography (LC) and Liquid Chromatography-Tandem Mass Spectrometry
(LC-MS/MS) etc..Wherein, the sensitivity of LC method is lower, can not confirm to doubtful positive sample；Although LC-MS/MS method,
Have many advantages, such as qualitative accurate, high sensitivity, but both for the detection of target compound, cannot achieve the sieve of a variety of colorants
It looks into, still will appear the detection of false positive sample, there is a problem of that detection efficiency is low.
Therefore, how to research and develop one kind can be achieved detection while a variety of colorants, to improve detection efficiency, become people urgently
Problem to be solved.
Summary of the invention
In consideration of it, the present invention provides a kind of method of acid stain in detection animal meat products, it is existing at least to solve
There is colorant detection method tested sample can only be caused to need to reprocess based on the detection of one or more of colorants,
It detects respectively, the problems such as time and effort consuming, detection efficiency is low.
Technical solution provided by the invention, specifically, a kind of method for detecting acid stain in animal meat products, the party
Method includes the following steps:
1) sample pre-treatments:
It weighs sample to be placed in centrifuge tube, after extracting solution is added, carries out mechanical shaking extraction, centrifugation, after collecting supernatant, then
Extracting solution is added and repeats to extract primary, collection supernatant, supernatant will merge twice；
By the supernatant after merging through WAX Solid Phase Extraction column purification, it is concentrated, after constant volume, it is spare；
2) qualitative analysis:
Screening is carried out to the animal meat products sample to be detected Jing Guo sample pre-treatments with UPLC-MS/MS-Qtrap method,
Using the enhancer ion scan function of liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer, MRM-IDA-EPI sieve is established
Checking method obtains the MS of each subject additives parent ion in animal meat products sample to be detected2Spectrogram；
With the standard MS of additive each in given data storehouse2Each target in spectrogram and acquisition animal meat products sample to be detected
The MS of additive parent ion2Spectrogram compares respectively, obtains similarity value, and matches the fragments characteristic ion of the two, in turn
Judge whether contain target acid stain in animal meat products sample to be detected；
3) quantitative analysis:
Each acid stain standard items are weighed respectively, prepare the standard reserving solution of 10 μ g/mL respectively with water, it is spare；
After sample-pretreating method handles blank sample in step 1), diluted with blank sample matrix solution
Hybrid standard stock solution is made in standard reserving solution；
After hybrid standard stock solution constant volume, obtain blank sample matrix solution, compound concentration be 0.05 μ g/mL~
The serial matching criteria working solution of 5.0 μ g/mL；
With UPLC-MS/MS-Qtrap method, using liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer to series
Matching criteria working solution and animal meat products sample to be detected Jing Guo sample pre-treatments are measured respectively, are obtained with concentration
For abscissa, the peak area of quota ion is the drafting standard curve of ordinate, calculates livestock and poultry to be detected with this standard curve
The content of target coloration agent in meat products sample.
It is preferred that the extracting solution is the mixed solution of methanol and ammonium hydroxide, wherein the volume ratio of methanol and ammonium hydroxide is 3:7, and
The volumetric concentration of ammonium hydroxide is 5%.
Further preferably, the acid stain are as follows: lemon yellow, sunset yellow, the red, newly red of temptation, famille rose, erythrosine,
One of acid red 73, azogeramine, acid orange 2 and azogeramine 14 are a variety of.
Further preferably, sample pre-treatments in the step 1) specifically:
5.00g sample is weighed, is placed in 100mL centrifuge tube, extracting solution 20mL, mechanical shaking extraction 20min, 8000r/ is added
Min is centrifuged 5min, and after collecting supernatant in another centrifuge tube, addition 20mL extracting solution repeats to extract primary again, in collection
Clear liquid supernatant will merge twice；
It takes supernatant 20mL to cross WAX solid-phase extraction column, is successively activated with 5mL methanol, 5mL water, then use 5mL 2% respectively
Formic acid, the elution of 5mL methanol, the elution of 5% ammonium hydroxide methanol solution of 6mL, 40 DEG C of nitrogen are blown to close dry, addition 1.0mL methanol-water (2:8)
Solution dissolved residue, sample liquid crosses 0.22 μm of filter membrane, spare.
Further preferably, in the step 2) liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer Mass Spectrometry Conditions
Are as follows:
Ion source: electric spray ion source；
Scan pattern: anion scanning；
Acquisition mode: multiple-reaction monitoring；
Information association scanning: intensity threshold 100cps；
Enhancer ion scan: 50~1000Da of mass number acquisition range m/z；
Electron spray voltage: -4500V；
Atomization gas pressure: 50psi；
Gas curtain atmospheric pressure: 30psi；
Assist gas pressure power: 50psi；
Ion source temperature: 450 DEG C；
Remove cluster voltage: -60V；
Entrance potential: -10V；
Collision cell exit potential: -10V；
Collision gas energy: 20,35,50V；
Extend collision energy: 15V.
Further preferably, in the step 3) liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer chromatographic condition
Are as follows:
Chromatographic column: Waters BEH C18 reverse-phase chromatographic column；
Column temperature: 40 DEG C；
Sampling volume: 10 μ L；
Flow velocity: 0.2mL/min；
Mobile phase: mobile phase A: 0.02mol/L ammonium acetate solution, B: methanol, gradient elution program: 0min, 90%A；
1min, 90%A；5min, 10%A；7min, 10%A；7.1min, 90%A；12min, 90%A；
The Mass Spectrometry Conditions of liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer in the step 3) are as follows:
Ion source: electric spray ion source；
Scan pattern: anion scanning；
Acquisition mode: multiple-reaction monitoring；
Electron spray voltage: -4500V；
Atomization gas pressure: 50psi；
Gas curtain atmospheric pressure: 30psi；
Assist gas pressure power: 50psi；
Ion source temperature: 450 DEG C；
Remove cluster voltage: -60V；
Entrance potential: -10V；
Collision cell exit potential: -10V.
Further preferably, the animal meat products sample to be detected is spiced beef and sausage.
The method of acid stain in detection animal meat products provided by the invention, this method are carried out to be checked using extracting solution
The extraction of sample, and by the supernatant obtained after extraction through WAX Solid Phase Extraction column purification, to complete sample pre-treatments, then
Using ultra performance liquid chromatography-series connection quadrupole rod linear ion trap mass spectrometer, 10 kinds of acidic colorants in animal meat products are established
Agent screening simultaneously, confirmation detection method, the detection method have easy to operate, flux is efficient, qualitative accurate, high sensitivity,
The features such as environmental-friendly, screening, confirmation suitable for acid stains a variety of in meat products can greatly improve detection effect
Rate avoids the detection of false positive sample.
It should be understood that above general description and following detailed description be only it is exemplary and explanatory, not
The disclosure can be limited.
Detailed description of the invention
The drawings herein are incorporated into the specification and forms part of this specification, and shows and meets implementation of the invention
Example, and be used to explain the principle of the present invention together with specification.
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, for those of ordinary skill in the art
Speech, without creative efforts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the MS that the present invention discloses a kind of doubtful sample of animal meat products to be detected that embodiment provides2Spectrogram；
Fig. 2 is that the present invention discloses the red standard MS of colorant-temptation that embodiment provides2Spectrogram.
Specific embodiment
Example embodiments are described in detail here, and the example is illustrated in the accompanying drawings.Following description is related to
When attached drawing, unless otherwise indicated, the same numbers in different drawings indicate the same or similar elements.Following exemplary embodiment
Described in embodiment do not represent all embodiments consistented with the present invention.On the contrary, they be only with it is such as appended
The example of method being described in detail in claims, some aspects of the invention are consistent.
In view of in the prior art, when carrying out colorant detection, can only be led based on the detection of one or more of colorants
The problems such as tested sample of cause needs to reprocess, and detects respectively, time and effort consuming, detection efficiency is low.The present embodiment passes through
The distinctive MRM-IDA-EPI screening function of UPLC-MS/MS Qtrap mass spectrum, and then realize 10 kinds of acid stains in meat products
While screening and confirmation, it is specific as follows:
One, sample pre-treatments:
1, sample extraction:
5.00g sample is accurately weighed, is placed in 100mL centrifuge tube, it is molten that -5% ammonium hydroxide of extracting solution methanol (3:7) mixing is added
Liquid 20mL, mechanical shaking extraction 20min, 8000r/min are centrifuged 5min, collect supernatant in another centrifuge tube, add 20mL and mention
It takes liquid to repeat to extract primary, collection supernatant, and supernatant will merge twice.
2, sample purification:
It takes 20mL supernatant to cross WAX solid-phase extraction column (successively with 5mL methanol, the activation of 5mL water), uses 5mL2% first respectively
Acid, the elution of 5mL methanol, the elution of 5% ammonium hydroxide methanol solution of 6mL, 40 DEG C of nitrogen, which are blown to, closely to be done, and it is molten that 1.0mL methanol-water (2:8) is added
Liquid dissolved residue, sample liquid crosses 0.22 μm of filter membrane, to be measured.
Two, qualitative analysis:
The legal measurement of UPLC-MS/MS-Qtrap: using MRM-IDA-EPI screening confirmation method, it can be achieved that qualitative, quantitative
It is disposable to complete, it can be applied to the analysis of complex matrices sample.
First use to be detected livestock meat of the liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer Jing Guo sample pre-treatments
Product sample carries out detection scanning, and whether system judges automatically the signal strength of detection scanning acquisition more than in IDA condition setting
Every setting value, that is, judge whether chromatographic peak occur.When meeting preset level, system quickly (< 1ms) automatically switches to line
Property ion trap mode, carry out enhancer ion scan (EPI), and obtain the high quality MS of parent ion obtained by detection scanning2Spectrogram,
With the standard MS of colorant each in given data storehouse2Each target coloration agent is female in spectrogram and acquisition animal meat products sample to be detected
The MS of ion2After spectrogram compares respectively, similarity value is obtained, and matches the fragments characteristic ion of the two, and then is judged
Whether contain target coloration agent in animal meat products sample to be detected.
Three, quantitative analysis:
It is measured using UPLC-MS/MS-Qtrap standard measure:
Matrix matching standard working solution and sample to be tested are measured, using concentration as abscissa, quota ion
Peak area is that ordinate draws standard curve, and the content of target coloration agent in test sample is calculated with this standard curve.
Wherein, the chromatographic condition that liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer uses in quantitative analysis are as follows:
Acquity Waters BEH C18 column (2.1 × 100mm, 1.7 μm), mobile phase A: 0.02% ammonium acetate solution；B: methanol,
Using gradient elution, condition of gradient elution is shown in Table 1, flow velocity 0.2mL/min, and 40 DEG C of column temperature, 10 μ L of sampling volume.
1. liquid chromatogram gradient elution program of table
| Time (min) | Flow velocity (mL/min) | Mobile phase | Mobile phase B % |
| 0 | 0.2 | A%90 | 10 |
| 1 | 0.2 | 90 | 10 |
| 5 | 0.2 | 10 | 90 |
| 7 | 0.2 | 10 | 90 |
| 7.1 | 0.2 | 90 | 10 |
| 12 | 0.2 | 90 | 10 |
Wherein, the Mass Spectrometry Conditions that liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer uses are as follows: ionization mode: electricity
Spraying anion ESI (-)；Scan pattern: anion scanning；Detection mode: multiple-reaction monitoring scans (MRM)；Information association is swept
It retouches: intensity threshold 100cps；Enhancer ion scan: 50~1000Da of mass number acquisition range m/z；Electron spray voltage
(IS): -4500V；Atomization gas pressure (GS1): 50psi；Gas curtain atmospheric pressure (CUR): 30psi；Assist gas pressure power (GS2):
50psi；Ion source temperature (TEM): 450 DEG C；Remove cluster voltage: -60V；Entrance potential (EP): -10V；Collision cell exit potential
(CXP): -10V；Collision gas energy: 20,35,50V；Extend collision energy: 15V.
The retention time and mass spectrum acquisition parameter of 10 kinds of colorants are shown in Table 2.
The retention time and mass spectrometry parameters of the colorant to be measured of table 2.
* quota ion.
The method of colorant compared with prior art, has following excellent in the detection animal meat products that the present embodiment provides
Good effect:
(1) the present embodiment establishes a kind of UPLC-MS/MS- for analyzing 10 kinds of acid stains in animal meat products
Qtrap screening, confirmation method, detection, confirmation while realizing a variety of colorants.
(2) the present embodiment is extracted using -5% ammonium hydroxide of methanol (3:7) mixed solution, the sample of MAX Solid Phase Extraction column purification
Product pre-treating method realizes 10 kinds of acid stains while extracting, purifying, and has easy, efficient, quick, environmental-friendly etc.
Advantage.
(3) the present embodiment is good using selectivity, and the UPLC-MSMS-Qtrap method detection of strong antijamming capability is stablized
Property good, high sensitivity, accuracy and precision meets the requirement of carbamate insecticides, while having the function of screening, confirmation,
It can avoid the detection of false positive sample.MRM-IDA-EPI function is selected, relevant criterion is set in IDA, connects liquid
Phase inputs the acquisition condition of liquid phase, carries out data acquisition, once the signal strength of MRM acquisition is more than criterion value, will touch
It sends out EPI and carries out enhancer ion scan acquisition MS2Figure, opens its suspicious peak (by retention time, peak shape etc. to the map of acquisition
Judgement) MS2Figure carries out the operations such as known spectrum library searching, compares with the mass spectrometric data for composing library, obtain corresponding coupling number
According to judge remaining detection, and then realizing the purpose of rapid screening and confirmation.
(4) detection limit of the present embodiment and quantitative limit are low, method high sensitivity, and the rate of recovery is stablized, and reproduction is acted charitably, met
The requirement of multi-residue analysis.
Specific embodiment
The present invention is further illustrated with specific embodiment below, but is not intended to restrict the invention
Protection scope.
Measurement of the above method to 10 kinds of acidic colorant agent contents in two kinds of sausage, spiced beef animal meat products is used below
And suspicious peak screening.
1. instrument and reagent
Key instrument: ACQUITYTM Ultra Performance Liquid Chromatography instrument (Waters, US)；AB Sciex QTrap
5500 quadrupole rods-ion trap mass spectrometer (American AB company).
Main agents: lemon yellow, sunset yellow lure red, newly red, famille rose, erythrosine, and purity >=99.0%, is purchased from
Country's mark object center；Acid red 73, azogeramine, acid orange 2, azogeramine 14, purity >=98.0%, are purchased from Germany
Dr.Ehrenstorfer。
2. standard solution is prepared:
Prepare standard reserving solution: weighing that each standard items are appropriate respectively, the standard reserving solution of about 10 μ g/mL is formulated as with water,
It is placed in 0 DEG C~4 DEG C refrigerators and saves backup.
Prepare matrix matching standard working solution: in order to eliminate influence of the matrix effect to quantitative determination, using blank sample
Solution dilutes hybrid standard stock solution.It is molten with blank sample matrix to blank sample by following identical pretreatment mode processing
Liquid dilutes hybrid standard stock solution, and constant volume obtains blank sample matrix solution, compound concentration is 0.05 μ g/mL, 0.2 μ g/mL,
The serial matching criteria working solution of 0.5 μ g/mL, 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL.
3. chromatograph mass spectrum analysis condition:
Chromatographiccondition:
Waters BEH C18 reverse-phase chromatographic column；Flow velocity: 0.2mL/min；Mobile phase A: 0.02mol/L ammonium acetate is water-soluble
Liquid, B: methanol；Using gradient elution, flow velocity 0.2mL/min, gradient elution program: 0min, 90%A；1min, 90%A；5min,
10%A；7min, 10%A；7.1min, 90%A；12min, 90%A；40 DEG C of column temperature；10 μ L of sampling volume.
Mass spectral analysis condition:
Ion source: electric spray ion source；Scan pattern: anion scanning；Acquisition mode: multiple-reaction monitoring (MRM)；EFI
Mist voltage (IS): -4500V；Atomization gas pressure (GS1): 50psi；Gas curtain atmospheric pressure (CUR): 30psi；Assist gas pressure power
(GS2): 50psi；Ion source temperature (TEM): 450 DEG C；Remove cluster voltage (DP): -60V；Entrance potential (EP): -10V；Collision cell
Exit potential (CXP): -10V.Wherein, the retention time and mass spectrum acquisition parameter of 10 kinds of colorants are shown in Table 2.
4. sample-pretreating method:
Sample extraction: accurately weighing 5.00g sample, is placed in 100mL centrifuge tube, and -5% ammonium hydroxide of extracting solution methanol is added
(3:7) mixed solution 20mL, mechanical shaking extraction 20min, 8000r/min are centrifuged 5min, collect supernatant in another centrifuge tube, then
20mL extracting solution is added and repeats to extract primary, combined extract.
Sample purification: it takes 20mL supernatant to cross WAX solid-phase extraction column (successively with 5mL methanol, the activation of 5mL water), uses respectively
2% formic acid of 5mL, the elution of 5mL methanol, the elution of 5% ammonium hydroxide methanol solution of 6mL, 40 DEG C of nitrogen are blown to close dry, addition 1.0mL methanol-
Water (2:8) solution dissolved residue, sample liquid crosses 0.22 μm of filter membrane, to be measured.
5. the efficiency evaluation of quantitative analysis and method:
Linearly, detection limit and quantitative limit: being measured the matrix matching standard solution of series of concentrations, with concentration for horizontal seat
Mark, the peak area of quota ion are that ordinate draws standard curve.Then test sample is measured, obtains various colorants
Quota ion peak area, substitute into standard curve, calculate 10 kinds of colorants content.Not homogeneity is added into blank sample
The standard solution of amount calculates separately out the detection limit and quantitative limit of method with 3 times of signal-to-noise ratio and 10 times of signal-to-noise ratio.10 kinds of colorants
Detection limit between 0.002~0.05mg/kg, between 0.007~0.165mg/kg, the linear of each compound returns quantitative limit
Equation, detection limit, quantitative limit is returned to be shown in Table 1.
Equation of linear regression, correlation coefficient r, the detection limit, quantitative limit of 1. colorant of table
Accuracy and precision: recovery testu, each concentration are done in tri- 0.2,0.4,2.0mg/kg concentration levels
Level does 6 parallel samples, carries out sample pre-treatments and instrument analysis respectively, and calculate recycling according to scalar quantity and measured value
Rate.The average recovery rate of 10 kinds of colorants and relative standard deviation (RSD) are shown in Table 2, table 3 in animal meat products.It can be with by table 2, table 3
Find out, average recovery rate of 10 kinds of colorants in two kinds of sausage, spiced beef animal meat products between 78.4%~97.7%,
Relative standard deviation meets the requirement of multi-residue analysis between 4.4%~14.3%.
The rate of recovery and relative standard deviation (n=6) in 2. sausage sample of table under different pitch-based spheres
The rate of recovery and relative standard deviation (n=6) in 3. spiced beef sample of table under different pitch-based spheres
6. suspicious peak qualitatively screening analysis citing
High-throughput unknown material rapid screening is carried out using Analyst software.Actual sample is detected, in MRM scan pattern
Lower discovery suspected target object, by comparing fragment, abundance ratio, guarantor in actual sample testing result and database under three kinds of energy
The information such as time are stayed to carry out compound confirmation.
Mass Spectrometry Conditions are as follows: ion source: electric spray ion source；Scan pattern: anion scanning；Acquisition mode: more reaction prisons
It surveys (MRM)；Information association scans (IDA): intensity threshold 100cps；Enhancer ion scan (EPI), mass number acquisition range m/
50~1000Da of z；Electron spray voltage (IS): -4500V；Atomization gas pressure (GS1): 50psi；Gas curtain atmospheric pressure (CUR):
30psi；Assist gas pressure power (GS2): 50psi；Ion source temperature (TEM): 450 DEG C；Remove cluster voltage (DP): -60V；Entrance potential
(EP): -10V；Collision cell exit potential (CXP): -10V；Collision gas energy (CE): 20,35,50V；Extend collision energy
(CES): 15V.
Select MS2Spectrogram and maximize figure, right click mouse clicks Search Library, and software can be given automatically
The result of screening out.Fit value (matching value) be obtained after being compared with standard substance spectrogram to suspicious specimen spectrogram it is similar
Angle value, full marks 100, the bigger confidence level of Fit value is higher, and a possibility that illustrating compound, is bigger；Revfit value (reverse phase matching value)
It is the similarity value obtained after being compared with suspicious specimen spectrogram and standard substance spectrogram；Purity value (Reinheitszahl) is comprehensive
The numerical value that first two result obtains.Fig. 1 is the MS at suspicious peak in sample2Spectrogram, Fig. 2 are the MS for luring red standard items2Spectrogram, tool
The spectrogram screening results of body are shown in Table 4.
Table 4. composes library screening results
From table 4, it is evident that luring red (Allura red) score high in library, arrangement near preceding, and fragments characteristic from
Son all with matched in sample, thus can determine whether in this sample containing lure it is red, further improve to result confirmation confidence, keep away
The generation of false positive results is exempted from.
Those skilled in the art after considering the specification and implementing the invention disclosed here, will readily occur to of the invention its
Its embodiment.This application is intended to cover any variations, uses, or adaptations of the invention, these modifications, purposes or
Person's adaptive change follows general principle of the invention and including the undocumented common knowledge in the art of the present invention
Or conventional techniques.The description and examples are only to be considered as illustrative, and true scope and spirit of the invention are wanted by right
It asks and points out.
It should be understood that the invention is not limited to the content being described above, can without departing from the scope into
Row various modifications and change.The scope of the present invention is limited only by the attached claims.
Claims (7)
1. a kind of method of acid stain in detection animal meat products, which comprises the steps of:
1) sample pre-treatments:
It weighs sample to be placed in centrifuge tube, after extracting solution is added, carries out mechanical shaking extraction, centrifugation adds after collecting supernatant
Extracting solution repeats to extract primary, collection supernatant, supernatant will merge twice；
By the supernatant after merging through WAX Solid Phase Extraction column purification, it is concentrated, after constant volume, it is spare；
2) qualitative analysis:
Screening is carried out to the animal meat products sample to be detected Jing Guo sample pre-treatments with UPLC-MS/MS-Qtrap method, is used
The enhancer ion scan function of liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer, establishes the screening side MRM-IDA-EPI
Method obtains the MS of each subject additives parent ion in animal meat products sample to be detected2Spectrogram；
With the standard MS of additive each in given data storehouse2Each target is added in spectrogram and acquisition animal meat products sample to be detected
The MS of agent parent ion2Spectrogram compares respectively, obtains similarity value, and matches the fragments characteristic ion of the two, and then judges
Whether contain target acid stain in animal meat products sample to be detected；
3) quantitative analysis:
Each acid stain standard items are weighed respectively, prepare the standard reserving solution of 10 μ g/mL respectively with water, it is spare；
After sample-pretreating method handles blank sample in step 1), with blank sample matrix solution dilution standard
Hybrid standard stock solution is made in stock solution；
After hybrid standard stock solution constant volume, blank sample matrix solution is obtained, compound concentration is 0.05 μ of μ g/mL~5.0 g/
The serial matching criteria working solution of mL；
With UPLC-MS/MS-Qtrap method, series is matched using liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer
Standard working solution and animal meat products sample to be detected Jing Guo sample pre-treatments are measured respectively, and obtaining with concentration is cross
Coordinate, the peak area of quota ion be the drafting standard curve of ordinate, calculates livestock meat system to be detected with this standard curve
The content of target coloration agent in product sample.
2. detecting the method for acid stain in animal meat products according to claim 1, which is characterized in that the extracting solution
For the mixed solution of methanol and ammonium hydroxide, wherein the volume ratio of methanol and ammonium hydroxide is 3:7, and the volumetric concentration of ammonium hydroxide is 5%.
3. detecting the method for acid stain in animal meat products according to claim 1, which is characterized in that the acidity
Toner are as follows: lemon yellow, lures red, newly red, famille rose, erythrosine, acid red 73, azogeramine, acid orange 2 and acid at sunset yellow
Property red one of 114 or a variety of.
4. detecting the method for acid stain in animal meat products according to claim 1, which is characterized in that the step 1)
Middle sample pre-treatments specifically:
Weigh 5.00g sample, be placed in 100mL centrifuge tube, be added extracting solution 20mL, mechanical shaking extraction 20min, 8000r/min from
Heart 5min after collecting supernatant in another centrifuge tube, 20mL extracting solution is added again and repeats to extract once, collects supernatant,
Supernatant it will merge twice；
It takes supernatant 20mL to cross WAX solid-phase extraction column, is successively activated with 5mL methanol, 5mL water, then use 2% first of 5mL respectively
Acid, the elution of 5mL methanol, the elution of 5% ammonium hydroxide methanol solution of 6mL, 40 DEG C of nitrogen, which are blown to, closely to be done, and it is molten that 1.0mL methanol-water (2:8) is added
Liquid dissolved residue, sample liquid crosses 0.22 μm of filter membrane, spare.
5. detecting the method for acid stain in animal meat products according to claim 1, which is characterized in that the step 2)
The Mass Spectrometry Conditions of middle liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer are as follows:
Ion source: electric spray ion source；
Scan pattern: anion scanning；
Acquisition mode: multiple-reaction monitoring；
Information association scanning: intensity threshold 100cps；
Enhancer ion scan: 50~1000Da of mass number acquisition range m/z；
Electron spray voltage: -4500V；
Atomization gas pressure: 50psi；
Gas curtain atmospheric pressure: 30psi；
Assist gas pressure power: 50psi；
Ion source temperature: 450 DEG C；
Remove cluster voltage: -60V；
Entrance potential: -10V；
Collision cell exit potential: -10V；
Collision gas energy: 20,35,50V；
Extend collision energy: 15V.
6. detecting the method for acid stain in animal meat products according to claim 1, which is characterized in that the step 3)
The chromatographic condition of middle liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer are as follows:
Chromatographic column: Waters BEH C18 reverse-phase chromatographic column；
Column temperature: 40 DEG C；
Sampling volume: 10 μ L；
Flow velocity: 0.2mL/min；
Mobile phase: mobile phase A: 0.02mol/L ammonium acetate solution, B: methanol, gradient elution program: 0min, 90%A；1min,
90%A；5min, 10%A；7min, 10%A；7.1min, 90%A；12min, 90%A；
The Mass Spectrometry Conditions of liquid chromatography-tandem quadrupole rod linear ion trap mass spectrometer in the step 3) are as follows:
Ion source: electric spray ion source；
Scan pattern: anion scanning；
Acquisition mode: multiple-reaction monitoring；
Electron spray voltage: -4500V；
Atomization gas pressure: 50psi；
Gas curtain atmospheric pressure: 30psi；
Assist gas pressure power: 50psi；
Ion source temperature: 450 DEG C；
Remove cluster voltage: -60V；
Entrance potential: -10V；
Collision cell exit potential: -10V.
7. detecting the method for acid stain in animal meat products according to claim 1, which is characterized in that described to be detected
Animal meat products sample is spiced beef and sausage.
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