CN106018583B - A kind of method of brilliant black and rhodamine B in LC-MS/MS measure food - Google Patents
A kind of method of brilliant black and rhodamine B in LC-MS/MS measure food Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of LC MS/MS to measure the method for brilliant black and rhodamine B in food.The present invention first crushes sample, heating water bath extracts, centrifuges, after supernatant tune acid, the Solid phase extraction pillar (cation exchange filler+anion exchange filler) of self assembly is recycled to carry out purified treatment to sample extracting solution, purification, the enrichment to brilliant black can be realized while chaff interferent is effectively removed, and using LC MS/MS quantitative determinations.This method is easy to operate, good purification, high sensitivity, favorable reproducibility, meets food additives analytical standard, has certain novelty and applicability.
Description
Technical field
The present invention relates to the assay method of artificial tanning agent in food a kind of, and in particular to brilliant black and Luo Dan in a kind of food
The Solid Phase Extraction of bright B/LC-MS/MS methods.
Background technology
Artificial synthesized colouring agent is the organic pigment produced by the method for chemical synthesis, mainly with virtues such as benzene, toluene, naphthalenes
Hydro carbons chemical products are raw material, are bound up by a series of organic reactions such as sulfonation, halogenation, azos.Its of low cost, color
Pool is bright-coloured, strong coloring force, performance are stablized, and is widely used in food production processing industry.But artificial synthesized colouring agent is most
For azo compound, excessive eat can have human body teratogenesis carcinogenesis.In recent years, in order to strengthen to eat synthetic coloring matter
Management, many countries have done thoroughgoing and painstaking research to the physicochemical properties and security of edible synthesized coloring matter, have clearly defined
Use kind, use scope and the usage amount of edible synthesized coloring matter.Food and Agricultural Organization of the United Nations defines brilliant black PN
INS151 use scopes in different food products matrix are 12~500mg/kg；European Union 94/36/EC and (EU) No 1274/2013
Rules and regulations " can use 151 brilliant black PN " of E, most ambassador in processing fish and aquatic products, including mollusk and Crustaceans
Dosage is 250mg/kg；Hong Kong food colour allows to use brilliant black using regulation Chapter 132H, does not provide to limit the quantity；The U.S.,
The states such as Canada, Japan and Korea S then forbid to use brilliant black and rhodamine B, China GB 2760-2014 in processed food《Food security
National standard food additives use standard》The colouring agent for ratifying to use also does not include brilliant black and rhodamine B (rose red b).
Brilliant black, Chinese food black 1, English name Brilliant black, molecular formula C28H17N5Na4O14S4, No. CAS：
2519-30-4, is mainly used as chemical dyestuff at present.Rhodamine B has strong fluorescence in the solution, glimmering as cell in laboratory
The industries such as light coloring agent, coloured glass, characteristic fireworks and firecrackers.Since brilliant black and rhodamine B are cheap, it is understood that there may be illegal business
The problem of people is added into food in violation of rules and regulations.In order to ensure the interests of consumer, it is necessary to be detected to it.
At present, the detection method both at home and abroad for brilliant black and rhodamine B mainly has high performance liquid chromatography.Xiao Hailong etc. is carried
Having gone out a kind of HPLC methods for measuring 18 kinds of synthetic coloring matters such as brilliant black in food, (Xiao Hailong, Tu Haiyun, wait reversed phase high performance liquids
Phase chromatography quickly measures 18 kinds of water-soluble synthetic coloring matter [J] Chinese Journal of Health Laboratory Technology, 2011,21 (2) in food:
264-266”)；Shao Shiping etc. with HPLC have detected 5 kinds of pigments such as brilliant black, and (Shao Shiping, Xi Xinglin, wait in beverages and candy 5 kinds
Detection [J] Food Sciences of illegal addition pigment, 2011,32 (4):189-192”)；Yang Jianlong etc. is surveyed at the same time with LC-MS/MS
Determine adulterated dyestuff scarlet 808 and rose red b in cinnabar, it is for mineral substrate, and pre-treatment purification method is in the publication
Do not have been reported that (Yang Jianlong, Tan Wen .LC-MS/MS measure content [J] of adulterated dyestuff scarlet 808 and rose red b in cinnabar at the same time
Chinese Journal of Modern Applied Pharmacy, 2015,8 (32):979-983).Liquid chromatography is artificial synthesized colouring agent detection application in food
Widest method, but supporting confirmation technology is a lack of, it can not meet the qualitative confirmation of object in the food substrate of complexity
It is required that.The LC-MS/MS methods of synthetic coloring matter, pertain only to colored parts agent and without before pervasive in the detection food reported
Treatment and purification means.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of LC-MS/MS to measure brilliant black and sieve in food
The method of red bright B.Extraction of this method to sample, SPE column purifications, LC-MS/MS quantitatively detect brilliant black and rhodamine B in food
Method studied, the results showed that this method high sensitivity, favorable reproducibility is easy to operate, can be widely applied in food
Brilliant black and the detection of rhodamine B.
The technical solution adopted in the present invention is：A kind of method of brilliant black and rhodamine B in LC-MS/MS measure food, its
It is characterized in that, comprises the following steps：
(1) prepare：Take representative sample comminution, stir evenly sealing mark, it is stand-by；
(2) extract：Step (1) sample is taken, is placed in centrifuge tube, adds ultra-pure water, is extracted in 50-70 DEG C of water bath sonicator
10-30min, 4000r/min centrifuges 5-15min in centrifuge, pipettes supernatant aqueous citric acid solution and debugs pH value to 3-
6, obtain solution to be clean；
(3) purify：
A, the assembling of solid phase extraction column：40-60 μm of cation exchange filler and 40-60 μm of anion exchange filler are taken,
It is filled in the polypropylene pillar that bottom is polyethylene sieve plate, filler upper end is fixed with polyethylene sieve plate again；
B, column activates：Above-mentioned solid phase extraction pillar is activated with the water of pH value 6-7, methanol successively；
C, sample-adding elution：Above-mentioned solution to be clean is shifted to solid phase extraction column, is drenched successively with the water of pH value 3-6, methanol
Wash SPE columns；
D, elute：Low vacuum dries up SPE columns, elutes component to be analyzed with ammoniated methanol and receives, collection liquid is in 50 DEG C of nitrogen
Air-blowing is done, and is dissolved in water, and 0.22 μm of filter membrane is crossed after vortex mixing, is analyzed for LC-MS/MS；
(4) detect：The sample to be tested that step (3) obtains is measured with liquid chromatography-tandem level Four bar mass spectrum.
Preferably, following steps are specifically included：
(1) prepare：Representative sample 500g is taken to crush, stir evenly, sealing mark is stand-by.
(2) extract：Step (1) sample 10g is taken, is placed in 50mL centrifuge tubes, 25mL ultra-pure waters are added, in 50-70 DEG C of water
Ultrasonic extraction 10-30min is bathed, 4000r/min centrifuges 5-15min in centrifuge, pipettes supernatant aqueous citric acid solution tune
PH value is tried to 3-6, obtains solution to be clean；
(3) purify：
A, the assembling of solid phase extraction column：Take 40-80mg 40-60 μm cation exchanges fillers and 40-80mg 40-60 μm
Anion exchange filler, is filled in the polypropylene pillar that bottom is polyethylene sieve plate, filler upper end is consolidated with polyethylene sieve plate again
It is fixed；
The cation exchange filler is to be bonded polystyrene divinyl benzene copolymer (PCX), benzene sulphur selected from benzene sulfonic acid
At least one of sour bonded silica gel (SCX), propane sulfonic acid bonded silica gel (PRS)；It is preferred that PCX fillers；
The anion exchange filler is selected from polyamide (PA), NH2Aminopropyl bonded silica gel, polyaminated polystyrene
At least one of divinylbenzene copolymer (PWAX) filler；It is preferred that PWAX fillers；
The cation exchange filler and the ratio of anion exchange filler are weight ratio 1:0.5~1:2；It is preferred that 1：1；
Further preferably, purification extraction column is voluntarily filled and presented using 60mgPCX+60mgPWAX/6mL to carry out at purification sample
Reason；
B, column activates：Above-mentioned solid phase extraction pillar is activated with the water of 4-8mL pH value 6-7,4-8mL methanol successively；
C, sample-adding elution：Above-mentioned solution 8-15mL to be clean is shifted to solid phase extraction column, successively with 4-8mL pH value 3-6
Water, 4-8mL methanol elution SPE columns；
D, elute：Low vacuum dries up SPE columns, is eluted with the ammoniated methanol (preferably 5% ammoniated methanol) of 4-8mL 3%-6%
Component to be analyzed simultaneously receives, and collection liquid is dried up in 50 DEG C of nitrogen, and 1.0mL is settled to water, and 0.22 μm of filter membrane is crossed after vortex mixing,
Analyzed for LC-MS/MS；
(4) detect：The sample to be tested that step (3) obtains is measured with liquid chromatography-tandem level Four bar mass spectrum.
Preferably, the mass spectrographic instrument parameter condition of liquid chromatography-tandem level Four bar is as follows in the step (4)：
Mass Spectrometry Conditions are：
Selective reaction monitoring parent ion, daughter ion and collision energy are：
Beneficial effects of the present invention：
The present invention studies the content detection of brilliant black in food and rhodamine B, and sample is through ultra-pure water water bath sonicator
After extraction, purified with self assembly solid phase extraction column, synchronously quantitative determined using LC-MS/MS.This method realizability property is different
Artificial synthesized colouring agent once cross column extracting purification, a chromatographic process quantitative analysis, easy to operate, high sensitivity, reproduction
Property is good, accuracy rate is higher.It can be widely applied to brilliant black and the accurate quantitative analysis of rhodamine B in complicated food substrate, operation letter
List, high sensitivity, favorable reproducibility.
The present invention using self assembly Solid phase extraction pillar to sample extracting solution carry out purified treatment, and using the moon from
Solid phase extraction column is made in the mode that sub- filler and cationic fillers load in mixture by a certain percentage, can effectively remove the same of chaff interferent
Purification, enrichments of the Shi Shixian to the artificial synthesized colouring agent of heterogeneity, method sensitivity, accuracy and precision meet food
Additive analysis standard, has certain novelty and applicability.Aforesaid way at the same time, compared to using two different solid phase extractions
The mode that column separately extracts is taken, saves time and cost.And self assembly pillar of the present invention, can be according to pigment content in sample
The particle diameter and dosage of appropriate adjustment filler, it is convenient to be applicable in.The present invention carries out Solid Phase Extraction using PWAX columns first, its absorption is held
Amount is big, and effect of extracting is good.
In the method for the present invention, brilliant black and rhodamine B be carried out at the same time the pre-treatments such as extraction, purification (brilliant black of the invention with
Rhodamine B is non-homologue, since the property of different material is different, during extraction, it is difficult to which all objects are all carried
Take entirely, still, method of the invention, while extract, purify, and brilliant black and rhodamine B extraction comparison are complete, in embodiment
The rate of recovery can illustrate this point), and once upper machine testing saves time and cost.
Brief description of the drawings
Fig. 1 is methanol+5mmol ammonium acetate solutions brilliant black and the MRM chromatograms of rhodamine B；
Fig. 2 brilliant blacks and rhodamine B characteristic ion are to chromatogram；
Brilliant black and the MRM chromatograms of rhodamine B in Fig. 3 barbecue flavoring samples；
Brilliant black and the MRM of rhodamine B are to chromatogram in Fig. 4 barbecue flavoring mark-on samples；
Fig. 5 is the analysis chromatogram (flavoring) of embodiment 1；
Fig. 6 is the analysis chromatogram (fruit drops) of embodiment 2.
Embodiment
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention
Content is not limited solely to the following examples, and embodiment is not construed as limiting the scope of the present invention.
1 instrument and reagent
Liquid Chromatography-Tandem Mass Spectrometry instrument：Aglient1200 high performance liquid chromatographs, API4000 mass spectrograph (American ABs
SCIEX)；MS3 vortex vortex mixers (IKA companies)；Thermostatic ultrasonic water-bath (IKA companies)；(Hitachi, Japan is public for high speed freezing centrifuge
Department)；Sensibility reciprocal is the assay balance (Mei Tele companies of Switzerland) of 0.1mg；Milli-Q water purifiors (Millipore companies of the U.S.)；N-
EVAP24 nitrogen evaporators (U.S.)；Range is 10-100 μ L, 100-1000 μ L liquid-transfering guns (Eppendof companies).
Brilliant black, rhodamine B standard items (self-control)；Methanol：Chromatographically pure (Merk companies)；Acetonitrile：Chromatographically pure (Merk companies)；
Formic acid：Chromatographically pure (Merk companies)；Ammonium acetate：Analyze pure；Citric acid：Analyze pure；Ammonium hydroxide：Analyze pure；Absolute ethyl alcohol：Analysis
It is pure；PWAX fillers：Agela Technologies companies；PCX fillers：Agela Technologies companies；SCX fillers：
Agela Technologies companies；PRS fillers：Agela Technologies companies；NH2 aminopropyl bonded silica gel fillers：
Agela Technologies companies；C18SPE extraction columns：Agela Technologies companies；Cation exchange (PCX) SPE
Extraction column：Agela Technologies companies；Polyamide (PA) SPE extraction columns：Agela Technologies companies.
2 standard solution are prepared
Brilliant black, rhodamine B colouring agent standard items 100.0mg are weighed, is dissolved with water, and constant volume is in 100mL brown volumetric flasks
In, up to 1000mg/L standard reserving solutions.Brown bottle be stored in -18 DEG C it is spare.
Take blank sample to be handled by said extracted and purification method, obtain bare substrate extraction and cleaning liquid.With the base
Series standard working solution is made in matter solution dilution standard deposit, carries out LC-MS/MS analyses.Using the concentration of standard working solution as
Abscissa, peak area are ordinate, draw dependent linear equation.
3 sample extractions and purification
3.1 prepare：
Representative sample 500g is taken to crush, stir evenly sealing mark, it is stand-by.
3.2 extraction：
Step (1) sample is taken, is placed in centrifuge tube, adds ultra-pure water, 10-30min is extracted in 50-70 DEG C of water bath sonicator,
4000r/min centrifuges 5-15min in centrifuge, pipettes supernatant aqueous citric acid solution and debugs pH value to 3-6, obtains to be clean
Solution；
3.3 purification：
3.3.1 the assembling of solid phase extraction column：40-80mg 40-60 μm cation exchanges fillers and 40- are weighed respectively
80mg 40-60 μm anion exchange fillers, be filled in bottom be polyethylene sieve plate polypropylene pillar in, filler upper end again with
Polyethylene sieve plate is fixed；
The cation exchange filler is to be bonded polystyrene divinyl benzene copolymer (PCX), benzene sulphur selected from benzene sulfonic acid
At least one of sour bonded silica gel (SCX), propane sulfonic acid bonded silica gel (PRS), the anion exchange filler are selected from third
At least one of base ethylenediamine (PSA), polyamide (PA), polyaminated polystyrene divinyl benzene copolymer (PWAX) filler；
The cation exchange filler and the ratio of anion exchange filler are weight ratio 1:0.5~1:2.
3.3.2 column activates：Above-mentioned solid phase extraction pillar is activated with the water of 4-8mL pH value 6-7,4-8mL methanol successively；
3.3.3 sample-adding elution：Above-mentioned liquid 8-15mL to be clean is shifted to solid phase extraction column, successively with 4-8mL pH value 3-
6 water, 4-8mL methanol elution SPE columns；
3.3.4 elution：Low vacuum dries up SPE columns, elutes component to be analyzed with the ammoniated methanol of 4-8mL 3%-6% and connects
Receive, collection liquid is dried up in 50 DEG C of nitrogen, and 1.0mL is settled to water, and 0.22 μm of filter membrane is crossed after vortex mixing, for LC-MS/MS points
Analysis.
4 detections
Sample to be tested is measured with liquid chromatography-tandem level Four bar mass spectrum, and instrument parameter condition see the table below 1, and selection is anti-
Parent ion, daughter ion and collision energy should be monitored and be shown in Table 2.
The 1 mass spectrographic instrument parameter condition of liquid chromatography-tandem level Four bar of table
2 brilliant black of table and the LC-MS/MS analytical parameters of rhodamine B
| Title | Retention time (min) | Parent ion | Daughter ion | Remove cluster voltage DP (v) | Impact energy CE (v) |
| Brilliant black | 7.74 | 778.0 | 291.0*,732.0 | -214 | - 55, -43 |
| Rhodamine B | 11.65 | 443.1 | 399.1*,355.0 | 100 | 58,80 |
5. results and discussion
The optimization of 5.1 chromatographic mass spectrometry conditions
5.1.1 the selection of mobile phase
Compare the aqueous formic acid of acetonitrile+0.1%, methanol+5mmol/L ammonium acetate solutions, the formic acid water of methanol+0.1%
Influence of the solution difference mobile phase to chromatogram.Research is found using the formic acid water of acetonitrile+0.1%, the formic acid water of methanol+0.1% as stream
Dynamic phase, object peak shape is poor, and ionization response is low.Methanol+5mmol/L ammonium acetate solutions are mobile phase, target compound
Chromatographic peak is symmetrically sharp, and preferable with food mesostroma separating effect, MRM chromatograms are as shown in Figure 1.
5.1.2 the optimization of mass spectroscopy condition
The standard solution for using concentration to be 500 μ g/L first is carried out parent ion in a manner of peristaltic pump continuous sample introduction and swept entirely
Retouch, determine the parent ion mass number of brilliant black and rhodamine B, 2 grades of collision scannings are then carried out with parent ion again, select two abundance
Compare high daughter ion as qualitative and quota ion.Optimize Mass Spectrometry Conditions at the same time, determine mass spectrum optimum condition, establish polyion
Reaction monitoring pattern.Figure below is the characteristic ion chromatography of rhodamine B under positive ion mode in Fig. 2；Upper figure is anion mould in Fig. 2
The characteristic ion chromatography of brilliant black under formula.
The elimination of 5.2 sample substrate effects
Electric spray ion source (ESI) is easily influenced by sample substrate.Find that sample substrate has suppression to ionization in experiment
Effect, different sample substrates have differences brilliant black and rhodamine B ionization inhibition level., should to eliminate sample substrate effect
Sample substrate effect is eliminated using blank sample extracting solution as the dilution of mark solution.
The selection of 5.3 purification SPE columns
The SPE purification extraction columns for being commonly used to do pigment have cation exchange (such as PCX) SPE columns, anion-exchange column (such as
PWAX, PA) SPE columns etc. made of SPE columns and C18 powder, there is C18 powder hydrophobic effect to have suction-operated to nonpolar component,
It is commonly used to remove impurity, to water miscible colouring agent almost without adsorption capacity.According to the difference of colouring agent acid-base property, filler has
The adsorbed target thing of selectivity, as PCX powder has object rhodamine B good absorption, but adsorbs other acid stains
Difference；PA powder, PWAX powder have acid stain good selective absorption.Since existing acidity also has alkali in 8 kinds of colouring agents
Property compound, this problem mainly study using once cross column solve purifying problem.Preferably, using PCX powder：When PWAX is filler
It is optimal to adsorb the compound ability of two kinds of properties, 60mgPCX：60mgPWAX/6mL voluntarily fills and presents purification extraction column and sample is carried out
Purified treatment.
The selection of 5.4 eluents
This research tests the SPE columns after pure methanol elution loading, it is found that target compound almost elutes to get off.Compare
1%th, elution effect of 2%, 4%, 5%, 6%, 10% ammoniated methanol as eluent.It was found that when equally taking 6mL eluents,
5% ammoniated methanol can elute completely, therefore using 5% ammoniated methanol as eluent.
5.5 method validation
5.5.1 the linear and detection limit of method
Blank sample is handled by said extracted and purification process, obtains bare substrate extraction and cleaning liquid.With the base
Brilliant black titer is diluted to 100 μ g/L by matter solution, 200 μ g/L, 400 μ g/L, 800 μ g/L, the series standard work of 1000 μ g/L
Make liquid, rhodamine B standard series:20 μ g/L, 40 μ g/L, 80 μ g/L, 160 μ g/L, 200 μ g/L；After LC/MS/MS is analyzed,
Standard curve and related coefficient is calculated, is shown in Table 3.Brilliant black and rhodamine B have well in linear scope as can be seen here
Linear relationship, related coefficient are respectively 0.9929 and 0.9961.
When the detection limit (LOD) of method is with least concentration appearance on blank sample diluted matrix standard curve, wins the confidence and make an uproar
Extension rate than S/N=3 and sample handling processes is calculated.Quantitative limit (LOQ) is bent with blank sample diluted matrix standard
During least concentration appearance on line, the extension rate of signal-to-noise ratio S/N=10 and sample handling processes is taken to be calculated.Specific data
It is shown in Table 3.
3 brilliant black of table and the linear equation of rhodamine B, the range of linearity, related coefficient and method detection limit
5.5.2 the rate of recovery and precision
To being added recovery experiment in barbecue flavoring and fruit drops, since rhodamine B response is high, addition concentration is
5、10、50μg/kg；Brilliant black addition concentration is respectively 50,100,200 μ g/kg.Standard solution is added after claiming sample, vortex mixing is put
Set to 0 and carry out sample treatment by above after .5h.Each level is repeated 6 times, and surveys precision.It the results are shown in Table 4.As it can be seen that brilliant black and Luo Dan
Bright B adds average recovery rate between 81.2%-96.0% in barbecue flavoring and fruit drops, relative standard deviation (RSD)
Respectively less than 6.0%.
4 brilliant black of table and the rate of recovery and Precision Experiment result of rhodamine B
The application of 5.6 methods
Barbecue flavoring sample, and the sample of standard solution is added, operated by the experimental procedure of " 3 sample extractions and purification "
After upper machine, measure chromatogram as shown in Figure 3 and Figure 4：
As can be seen that barbecue flavoring sample substrate does not disturb the measure of brilliant black and rhodamine B, mark-on sample from chromatogram
Product recycling is preferable.But rhodamine B has background, it is necessary to detain blank in blank.
Experiment shows, sample extracting solution can be carried out using this law at purification using the Solid phase extraction pillar of self assembly
Reason, can realize purification, enrichment to brilliant black and rhodamine B while chaff interferent is effectively removed, and be quantified using LC-MS/MS
Measure, method sensitivity, accuracy and precision meet food additives analytical standard, fully meet the production of import and export food
Quality control requires.
Embodiment 1：
The measure of brilliant black and rhodamine B in barbecue flavoring sample.
3 sample extractions and purification
3.1 extraction
Representative sample 500g is taken to crush, stir evenly, sealing mark is stand-by.
It is placed in centrifuge tube, adds ultra-pure water, 20min, the 4000r/ in centrifuge is extracted in 50-70 DEG C of water bath sonicator
Min centrifuges 10min, pipettes supernatant aqueous citric acid solution debugging pH value 4, obtains solution to be clean；
3.2 purification
3.2.1 the assembling of solid phase extraction column (6mL)：Weigh respectively 60mg 40-60 μm PCX cation exchanges fillers and
60mg 40-60 μm PWAX mixed type weak anion-exchanges, are filled in the polypropylene pillar that bottom is polyethylene sieve plate,
Filler upper end is fixed with polyethylene sieve plate again；
3.2.2 column activates：Above-mentioned solid phase extraction pillar is activated with the water of 6mL pH value 6-7,6mL methanol successively；
3.2.3 sample-adding elution：Above-mentioned liquid 10mL to be clean is shifted to solid phase extraction column, successively with the water of 6mL pH value 4,
6mL methanol elutes SPE columns；
3.2.4 elution：Low vacuum dries up SPE columns, elutes component to be analyzed with the ammoniated methanol of 6mL 5% and receives, receives
Liquid collecting is dried up in 50 DEG C of nitrogen, and 1.0mL is settled to water, and 0.22 μm of filter membrane is crossed after vortex mixing, is analyzed for LC-MS/MS；
4 detections
Sample to be tested is measured with liquid chromatography-tandem level Four bar mass spectrum, and instrument parameter condition is shown in Table 1, selection reaction
Monitoring parent ion, daughter ion and collision energy are shown in Table 2；It analyzes chromatogram as shown in figure 5, the sample recovery rate and precision are real
Test and the results are shown in Table 4.
Embodiment 2
The measure of brilliant black and rhodamine B in fruit drops sample.
3 sample extractions and purification
3.1 extraction
Representative sample 500g is taken to crush, stir evenly sealing mark, it is stand-by.
It is placed in centrifuge tube, adds ultra-pure water, 15min, the 4000r/ in centrifuge is extracted in 50-70 DEG C of water bath sonicator
Min centrifuges 10min, pipettes supernatant aqueous citric acid solution and debugs pH value to 3-6, obtains solution to be clean；
3.2 purification
3.2.1 the assembling of solid phase extraction column (6mL)：Weigh respectively 60mg 40-60 μm PCX cation exchanges fillers and
60mg 40-60 μm PWAX mixed type weak anion-exchanges, are filled in the polypropylene pillar that bottom is polyethylene sieve plate,
Filler upper end is fixed with polyethylene sieve plate again；
3.2.2 column activates：Above-mentioned solid phase extraction pillar is activated with the water of 6mL pH value 6-7,6mL methanol successively；
3.2.3 sample-adding elution：Above-mentioned liquid 10mL to be clean is shifted to solid phase extraction column, successively with the water of 6mL pH value 4,
6mL methanol elutes SPE columns；
3.2.4 elution：Low vacuum dries up SPE columns, elutes component to be analyzed with the ammoniated methanol of 6mL 5% and receives, receives
Liquid collecting is dried up in 50 DEG C of nitrogen, and 1.0mL is settled to water, and 0.22 μm of filter membrane is crossed after vortex mixing, is analyzed for LC-MS/MS.
4 detections
Sample to be tested is measured with liquid chromatography-tandem level Four bar mass spectrum, and instrument parameter condition is shown in Table 1, selection reaction
Monitoring parent ion, daughter ion and collision energy are shown in Table 2；It analyzes chromatogram as shown in fig. 6, the sample recovery rate and precision are real
Test and the results are shown in Table 4.
Claims (5)
1. a kind of method of brilliant black and rhodamine B in LC-MS/MS measure food, it is characterized in that, comprise the following steps：
(1) prepare：Take representative sample comminution, stir evenly, sealing mark is stand-by；
(2) extract：Step (1) sample is taken, is placed in centrifuge tube, adds ultra-pure water, is extracted in 50-70 DEG C of water bath sonicator, centrifugation,
Pipette supernatant aqueous citric acid solution and debug pH value to 3-6, obtain solution to be clean；
(3) purify：
A, the assembling of solid phase extraction column：Take 40-60 μm of benzene sulfonic acid bonding polystyrene divinyl benzene copolymer p CX fillers and more
Amino polystyrene divinylbenzene copolymer PWAX fillers, are filled in the polypropylene pillar that bottom is polyethylene sieve plate, filler
Upper end is fixed with polyethylene sieve plate again；
The benzene sulfonic acid bonding polystyrene divinyl benzene copolymer p CX fillers and the copolymerization of polyaminated polystyrene divinyl benzene
The ratio of thing PWAX fillers is weight ratio 1:0.5~1:2；
B, column activates：Above-mentioned solid phase extraction pillar is activated with the water of pH value 6-7, methanol successively；
C, sample-adding elution：The solution to be clean of transfer step (2) is drenched with the water of pH value 3-6, methanol successively to solid phase extraction column
Wash solid phase extraction column；
D, elute：Solid phase extraction column is dried up, component to be analyzed is eluted with ammoniated methanol and receives, the drying of collection liquid nitrogen, adds
Water dissolves, and 0.22 μm of filter membrane is crossed after vortex mixing, is analyzed for LC-MS/MS；
(4) detect：The sample to be tested that step (3) obtains is measured with LC-MS/MS；
The instrument parameter condition of the LC-MS/MS is as follows：
Mass Spectrometry Conditions are：
2. the method for brilliant black and rhodamine B in a kind of LC-MS/MS measure food as claimed in claim 1, it is characterized in that, it is bright
Black and rhodamine B LC-MS/MS analytical parameters are as shown in the table：
3. the method for brilliant black and rhodamine B in a kind of LC-MS/MS measure food as claimed in claim 1 or 2, it is characterized in that,
(1) prepare：Take representative sample comminution, stir evenly, sealing mark is stand-by；
(2) extract：Step (1) sample 10g is taken, is placed in 50mL centrifuge tubes, 25mL ultra-pure waters is added, surpasses in 50-70 DEG C of water-bath
Sound extracts 10-30min, and 4000r/min centrifuges 5-15min in centrifuge, pipettes supernatant aqueous citric acid solution debugging pH
Value obtains solution to be clean to 3-6；
(3) purify：
A, the assembling of solid phase extraction column：Take 40-80mg 40-60 μm cation exchanges fillers and 40-80mg 40-60 μm the moon from
Son exchanges filler, is filled in the polypropylene pillar that bottom is polyethylene sieve plate, filler upper end is fixed with polyethylene sieve plate again；
B, column activates：Above-mentioned solid phase extraction pillar is activated with the water of 4-8mL pH value 6-7,4-8mL methanol successively；
C, sample-adding elution：The solution 8-15mL to be clean of transfer step (2) is to solid phase extraction column, successively with 4-8mL pH value 3-
6 water, 4-8mL methanol elution solid phase extraction column；
D, elute：Low vacuum dries up solid phase extraction column, elutes component to be analyzed with the ammoniated methanol of 4-8mL 3%-6% and connects
Receive, collection liquid is dried up in 50 DEG C of nitrogen, and 1.0mL is settled to water, and 0.22 μm of filter membrane is crossed after vortex mixing, for LC-MS/MS points
Analysis；
(4) detect：The sample to be tested that step (3) obtains is measured with LC-MS/MS.
4. the method for brilliant black and rhodamine B in a kind of LC-MS/MS measure food as claimed in claim 3, it is characterized in that, point
Also known as take 60mg 40-60 μm benzene sulfonic acids bonding polystyrene divinyl benzene copolymer p CX fillers and 60mg 40-60 μm polyaminated
Polystyrene divinyl benzene copolymer p WAX fillers.
5. the method for brilliant black and rhodamine B in a kind of LC-MS/MS measure food as claimed in claim 3, it is characterized in that, institute
State elution step and use 5% ammoniated methanol.
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