CN108303481A - The universal test method of citrus red2 number and Sudan red dyes in food - Google Patents
The universal test method of citrus red2 number and Sudan red dyes in food Download PDFInfo
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Abstract
The invention discloses the universal test method of citrus red2 number and Sudan red dyes in a kind of food, it is adapted to the detection of citrus red2 number and Sudan red dyes (including I, II, III, IV type) in numerous food matrix.By being studied respectively for the big food substrate of oil content, water content difference, acetonitrile sodium chloride the pre-treating method that system, amino Solid Phase Extraction column purification, formic acid acetonitrile redissolve is extracted for the first time to handle for being compatible with high grease and high-moisture matrix sample, the detection of the different food products matrix such as citrus fruit, jam, preserved fruit, fruit drink, chilli oil, thick chilli sauce, chafing dish bottom flavorings, meat products is adapted it to, pre-treatment operational suitability is wide, tolerance is good.Instrument accuracy height, high sensitivity, operability are strong, and detection efficiency is high.
Description
Technical field
The present invention relates to citrus red2s number in technical field of food detection more particularly to a kind of food and Sudan red dyes
Universal test method.
Background technology
Citrus red2 number is Chinese red powder, not soluble in water, is dissolved in aromatic hydrocarbon solvent, nearly 2 years, citrus red2 conduct was new
Emerging dyestuff, have increase fruit color and luster and the uniformity the effect of, citrus fruit after artificial dyestuff citrus red2 beauty on
City is sold, and improves the market competitiveness of fruit.
The World Health Organization thinks that citrus red2 number has carcinogenic danger, and the dyestuff is by European Union and Japan and other countries and area
It is disabled, Food and Drug Adminstration of the US's clear stipulaties：Through the dissolved citrus red2 number of suitable solvent, only limit the use of in being not intended to
The precocious hyperchromic use of sweet orange pericarp of processing, and consequences for use reality epidermis must not residual volatile solvent, citrus red2 in full fruit
Number maximum residue limit is no more than 2mg/kg.On October 27th, 2017, international cancer research institution of the World Health Organization discloses cause
Cancer object edit inventory, citrus red2 number is in 2B class carcinogen inventories.China's national standard《Food additives use is defended
Raw standard》It can be used for that citrus fruit is hyperchromic not to make regulation also to citrus red2 dyestuff, also not formulate mandarin orange in citrus fruit
No. 2 residual quantity standards of Exocarpium Citri Rubrum.
Tonyred is a kind of chemical dye, including I, II, III, IV 4 types.Be mainly used for oil, machine oil and other
Some industrial solvents in, it is therefore an objective to keep its hyperchromic.Once caused " red-yolk duck egg ", the food safety affairs such as " Heng Shi thick chilli sauce ".
Why will be added in food as the tonyred of industrial chemicals, especially makes in being processed with capsicum product：When by
Do not allow fugitive color after tonyred use, capsicum can be made up in this way and place the phenomenon that changing colour long afterwards, the color and luster for keeping capsicum vivid；
Second is that some enterprises are mixed in after the plant powders such as corn the Sudan's red colouring in chilli powder, are sought interests with reducing cost.
It is determined according to international cancer research institution's (IARC) nineteen ninety-five of the World Health Organization, tonyred category third class carcinogenic substance
Matter；European Union member countries forbid it as food coloring in nineteen ninety-five, and China exists《Food additives use sanitary standard》In also prohibit
Only tonyred is used as food additives.
The detection method of detection citrus red2 number and Sudan red dyes has at present《GB/T 19681-2005 Detection of Magdala in Food Through
The detection method high performance liquid chromatography of dyestuff》、《DB31/T 441-2009 Sudan I in foods, II, III, IV and para red
Measurement》、《The measurement high performance liquid chromatography of No. 2 dyestuffs of Exocarpium Citri Rubrum in DB51/T 1883-2014 citruses》、《DBS22/009-
The measurement high performance liquid chromatography of Exocarpium Citri Rubrum 2 in 2013 food security provincial standard citrus fruits and its beverage》The above method
Poor for applicability, matrix interference is serious, and resolution ratio and sensitivity are relatively low, and the detection that cannot be satisfied qualitative, quantitative in trace detection is wanted
It asks.
Method of the report for illegal dyestuff citrus red2 number and tonyred (including I, II, III, IV type) pre-treatment both at home and abroad
Main two kinds：Direct extraction method and the aluminium oxide method of purification.Two kinds of pretreatment modes are relatively preferable for the effect of single-matrix, but
It is undesirable for a variety of matrix extraction effects of different food products.
Report is less for citrus red2 number and Sudan red dyes (including I, II, III, IV type) detection method both at home and abroad, has
Thin-layered chromatography, gas chromatography-mass spectrography, high performance liquid chromatography, liquid chromatogram-level four bars time-of-flight mass spectrometry screening
Method, ultra high efficiency liquid phase-tandem mass spectrometry.Wherein thin-layered chromatography is complex for operation step, and quantitative inaccurate；Gas chromatography-mass spectrum
Method needs to carry out complicated derivatization process；High performance liquid chromatography is only adapted to detect while 5 kinds of dyestuffs in fraction matrix,
Detection sensitivity is relatively low；Liquid chromatogram-level four bars time-of-flight mass spectrometry screening technology is qualitative accurate, quantitative slightly worse, and
It is expensive, height is required to operating personnel's technology, for general testing agency's heavy load.In the document reported at present simultaneously
Ultra high efficiency liquid phase-tandem mass spectrometry of citrus red2 number and Sudan red dyes (including I, II, III, IV type) is detected just for capsicum
Product and meat products, narrow scope of application.
Invention content
The present invention provides the general inspections of citrus red2 number in a kind of food and tonyred (including I, II, III, IV type) dyestuff
Survey method, to solve the problems, such as to solve 5 kinds of dyestuffs while accurate qualitative and quantitative detection in numerous food matrix.
The universal test method of citrus red2 number and Sudan red dyes, includes the following steps in food provided by the invention：
(1) it prepares extraction standard solution and carries out high performance liquid chromatography-tandem mass detection
The Standard Stock solutions of the dyestuff of required test are diluted to the standard working solution of various concentration gradient, several
Standard working solution is added in vehicle solution known to part, obtains the blank mark-on sample of various concentration gradient.
(2) extraction standard solution-mass chromatogram and equation of linear regression are made
After blank mark-on sample is carried out pre-treatment, extraction standard solution is obtained；By extraction standard solution according to concentration ladder
Degree difference sample introduction, is detected with ultra performance liquid chromatography-tandem mass spectrum combined instrument, obtains extraction standard solution-mass chromatography
Figure；Using the extraction standard chromatographic peak area of standard quality chromatogram as ordinate, in extraction standard solution each dyestuff it is corresponding
Concentration value is mapped for abscissa, obtains standard curve and corresponding equation of linear regression.
(3) qualitative analysis of sample to be tested
By food substrate to be measured after pre-treatment, sample to be tested is obtained；With ultra performance liquid chromatography-tandem mass spectrum combination
Instrument detects sample to be tested, obtains sample to be tested-mass chromatogram, passes through retention time and the reference colour spectral peak of sample chromatographic peak
Retention time compares, and the relative abundance ratio of sample chromatographic peak determines contained dyestuff in sample compared with reference colour spectral peak
Type；Wherein, the reference colour spectral peak be by the standard working solution ultra performance liquid chromatography-tandem mass spectrum combined instrument into
Row detects, and is obtained in obtained standard items-mass chromatogram.
(4) the quantitative calculating of sample to be tested
Using quantified by external standard method：It is calculated using the equation of linear regression of step (2) according to the area of sample chromatographic peak
To the content of dyestuff in sample.
Wherein, dyestuff includes citrus red2 number, Sudan red type, Sudan II type, red Ⅲ type and SudanⅣ type；Step
Suddenly the pre-treatment in (2) and (3) is that sample is first passed through acetonitrile-sodium chloride system to extract, and takes out the drying of acetonitrile layer nitrogen, uses second
Nitrile-dichloromethane solution dissolving is redissolved using amino Solid Phase Extraction column purification with formic acid-acetonitrile solution, after crossing film, for super
High performance liquid chromatography-tandem mass combined instrument is detected, and the sample includes blank mark-on sample and food substrate to be measured；Institute
State the food substrate that food substrate to be measured includes high grease and/or high-moisture.
Optionally, in the pre-treatment, in acetonitrile-sodium chloride system, the ratio of acetonitrile-sodium chloride system and sample is
10mL:2g。
Optionally, the parameter of the ultra performance liquid chromatography-tandem mass spectrum combined instrument is as follows：
Liquid chromatogram parameter：
Liquid-phase chromatographic column：C18 liquid-phase chromatographic columns, column length 75mm, column internal diameter 2.1mm, 1.7 μm of filler particle size；
Column temperature：40℃；
Sampling volume：2.0μL
Mobile phase and gradient elution program：0-3min：0.1% aqueous formic acid 90% → 50%, acetonitrile 10% → 50%；
3.0-5.0min：0.1% aqueous formic acid 50% → 5%, acetonitrile 50% → 95%；5.0-7.0min：0.1% aqueous formic acid
5%, acetonitrile 95% keeps 2min；7.0-7.1min：0.1% aqueous formic acid 5% → 90%, acetonitrile 95% → 10%；7.1-
9.0min：0.1% aqueous formic acid 90%, acetonitrile 10%；
Mass spectrometry parameters：
Capillary voltage：3.0KV；Orifice potential：25V；Desolvation temperature：500℃；Ion source temperature：150℃；It is de-
Solvent stream speed：850L/Hr；Taper hole gas velocity：150L/Hr；Collision gas flow velocity：0.12mL/min；Scan mode：More reaction prisons
Survey MRM.
Optionally, the pre-treatment, includes the following steps：
It weighs 2g samples and 10mL acetonitriles is added in 50mL centrifuge tubes, ultrasonic 20min is added 2g sodium chloride, acutely shakes
1min, ultrasonic 10min centrifuge 3min with 8000r/min rotating speeds, take out in acetonitrile layer to 15mL centrifuge tubes, under 40 DEG C of nitrogen streams
Drying is dissolved with 3mL acetonitrile-dichloromethane solution, to be clean；
Liquid to be clean is shifted when liquid level is down to column bed with 6mL acetonitrile-dichloromethanes activation amino solid-phase extraction column
Into amino solid-phase extraction column, it is collected simultaneously scavenging solution；Twice with 3mL acetonitrile-dichloromethane rinse 15mL centrifuge tubes, continue to collect
Scavenging solution；It is concentrated to dryness under 40 DEG C of nitrogen streams, accurate 10mL formic acid-acetonitrile solution of drawing redissolves, after crossing miillpore filter, for liquid phase
Chromatography-tandem mass spectrometry measures.
Optionally, the food substrate to be measured includes fruit, jam, preserved fruit, fruit drink, chilli oil, thick chilli sauce, chafing dish
Bottom material or meat products.
Optionally, blank mark-on sample be each dye content be 5 μ g/kg, 10 μ g/kg, 50 μ g/kg, 100 μ g/kg,
The blank mark-on sample of 200 μ g/kg.
The invention has the advantages that：
(1) the general detection method of numerous food matrix
The present invention is adapted to the inspection of citrus red2 number and Sudan red dyes (including I, II, III, IV type) in numerous food matrix
It surveys.This patent is for the first time extracted acetonitrile-sodium chloride by being studied respectively for the big food substrate of oil content, water content difference
The pre-treating method that system, amino Solid Phase Extraction column purification, formic acid-acetonitrile redissolve is for being compatible with high grease and high-moisture matrix
Sample treatment adapts it to citrus fruit, jam, preserved fruit, fruit drink, chilli oil, thick chilli sauce, chafing dish bottom flavorings, meat products
The detection of equal different food products matrix, pre-treatment operational suitability is wide, tolerance is good.If carrying out mass detection, method adapts to model
Advantage wide, that tolerance is good is enclosed to will be apparent from.
1.1) acetonitrile-sodium chloride for being suitable for a variety of matrix extracts system
In view of the lipophilicity feature of citrus red2 number and Sudan red dyes (including I, II, III, IV type), for water content
High sample is extracted using acetonitrile, in addition sodium chloride is saltoutd, can effectively extract 5 kinds of illegal dyes in high-water content sample
Material；For containing the high sample of grease, being extracted with acetonitrile, impurity can be effectively removed, coextraction object is less.This method uses second
Nitrile-sodium chloride extracts system, and the extraction of 5 kinds of illegal dyestuffs suitable for different substrates, easy to operate, applicability is good.
1.2) it is suitable for the nh 2 column purification condition of a variety of matrix
The extraction of acetone-n-hexane is taken in terrestrial reference standard, does not take effective purification means, it can be with for fruit such as citruses
It adapts to, for the high oil sample such as thick chilli sauce, does not purify means, matrix interference is very serious, influences quantitative.Document has been reported that
The purification style of neutral alumina column, neutral alumina column needs to control the activity of aluminium oxide, complex for operation step, and chaff interferent
It is more.The present invention is unified to utilize amino solid-phase extraction column by being studied respectively the water content matrix different with oil content
Purification, chaff interferent is few, and matrix effect is small, meets the purification of the big sample of matrix components difference.
1.3) formic acid-acetonitrile for being suitable for a variety of matrix redissolves liquid
For high-water content matrix, after purified treatment, redissolved with acetonitrile or acetonitrile solution, the rate of recovery of object
No significant difference；For the high matrix of oil content, after purified treatment, however it remains a small amount of grease, if with acetonitrile or acetonitrile water
Solution redissolves, and object cannot be completely dissolved, and cause the rate of recovery relatively low.This method selects the first being mutually applicable in purification style
Acid-acetonitrile (1%) solution redissolves, and object has better choice, the rate of recovery good.
(2) instrument accuracy height, high sensitivity, operability are strong
Both at home and abroad report for citrus red2 number and Sudan red dyes Simultaneous Detection it is less, have thin-layered chromatography, efficiently
Liquid chromatography, ultra high efficiency liquid phase-tandem mass spectrometry, gas chromatography-mass spectrography, liquid chromatogram-level four bars are connected the flight time
Mass spectrum screening.Wherein thin-layered chromatography is complex for operation step, and quantitative inaccurate；Gas chromatography-mass spectrography needs to carry out complexity
Derivatization process, testing result precision are poor；High performance liquid chromatography is suitable for the discrete phases pair such as citrus fruit, beverage, ham
It is detected while 5 kinds of dyestuffs in simple food, but when for the sample detection of the matrix such as chafing dish bottom flavorings, chilli oil complexity, it is clever
Sensitivity is low, qualitative inaccuracy；Liquid chromatogram-level four bars time-of-flight mass spectrometry screening technology is qualitative accurate, quantitative slightly worse, and
And it is expensive, the country only has a small number of laboratories above the provincial level to possess the instrument, and requires height to operating personnel's technology, for
General testing agency's heavy load.In the document reported at present at the same detect citrus red2 number and Sudan red dyes (including I,
II, III, IV type) ultra high efficiency liquid phase-tandem mass spectrometry just for chilli products and meat products, narrow scope of application；For more
The method of 5 kinds of illegal dyestuffs detection simultaneously does not find to report in kind matrix, and this patent uses ultra high efficiency liquid phase-tandem mass spectrum for the first time
Method detects 5 kinds of illegal dyestuffs in numerous food matrix simultaneously, and accuracy is high, and high sensitivity meets the needs of general testing agency.
The detection of citrus red2 number and Sudan red dyes is limited to 5 μ g/kg in the present invention, is quantitatively limited to 10 μ g/kg, fully meets existing mark
Requirement of the standard to citrus red2 number and Sudan red dyes (including including I, II, III, IV type), sensitivity are not less than existing literature report
Road is horizontal, and it is horizontal that detection limit is even lower than part document detection limit.
(3) detection efficiency improves 5 times
Citrus red2 number and 5 kinds of compounds of tonyred (including I, II, III, IV type) are merged into primary experiment by the present invention, are examined
The survey time shortens 5 times.《The detection method high performance liquid chromatography of GB/T 19681-2005 Detection of Magdala in Food Through dyestuffs》Instrument
Analysis time is 30min, and the pre-treatment time is about 2h,《The measurement of No. 2 dyestuffs of Exocarpium Citri Rubrum is efficient in DB51/T 1883-2014 citruses
Liquid chromatography》The Instrumental Analysis time is 30min, and the pre-treatment time is about 2h, and 5 kinds of illegal dyestuff detection times need altogether
600min, instrument detection time of the invention are only 9min, and the pre-treatment time is about 2h, and the illegal dyestuff of 5 kinds of simple sample is simultaneously
The time of detection is only 129min, and detection time shortens nearly 5 times than above-mentioned two standard.If multiple types sample detects simultaneously,
Because pre-treatment can be carried out at the same time, the time can also substantially shorten, and the method for inspection of existing literature need to be directed to different matrix point
Class processing, detection efficiency decline.Integrated comparative finds that detection time of the invention is short, and detection efficiency is high.
It should be understood that above general description and following detailed description is only exemplary and explanatory, not
It can the limitation present invention.
Description of the drawings
In order to illustrate more clearly of technical scheme of the present invention, letter will be made to attached drawing needed in the embodiment below
Singly introduce, it should be apparent that, for those of ordinary skills, without having to pay creative labor,
Other drawings may also be obtained based on these drawings.
Fig. 1 citrus red2s number (10ng/mL) and tonyred (including I, II, III, IV type) (10ng/mL) standard items-quality color
Spectrogram；
Bare substrate-quality color of Fig. 2 bare substrates (without citrus red2 number and tonyred (including I, II, III, IV type))
Spectrogram；
Fig. 3 bare substrates mark-on (containing a concentration of 25 μ g/kg citrus red2s number and a concentration of 25 μ g/kg tonyreds (including I,
II, III, IV type)) extraction standard solution-mass chromatogram.
Wherein, sudanhong1, sudanhong2, sudanhong3, sudanhong4 be represented sequentially as Sudan red type,
Sudan II type, red Ⅲ type, SudanⅣ type；GJH2 indicates citrus red2 number.
Specific implementation mode
The universal test method of citrus red2 number and tonyred (including I, II, III, IV type) dyestuff in a kind of food, with solution
Certainly solve the problems, such as 5 kinds of dyestuffs while accurate qualitative and quantitative detection in numerous food matrix.Below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is only
It is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
The every other embodiment that personnel are obtained without making creative work, shall fall within the protection scope of the present invention.
Embodiment 1
1, instrument and material
Ultra performance liquid chromatography-tandem mass spectrometer, equipped with electron spray (ESI) ionization source, (XEVO TQ-S U.S. Waters are public
Department)；Chromatographic column：Acquity UPLC BEH C18 columns (75mm × 2.1mm i.d, 1.7 μm), (Waters, US).
Citrus red2 standard substance (purity >=90.0%) is purchased from German Dr.Ehrenstorfer companies；Tonyred (packet
Include I, II, III, IV type) standard substance (purity >=99.0%), it is purchased from German Dr.Ehrenstorfer companies；Acetonitrile (chromatography
It is pure)；N-hexane (chromatographically pure)；Dichloromethane (chromatographically pure)；Formic acid (chromatographically pure)；Sodium chloride (analysis is pure).Ultra-pure water：Mili-Q
It is prepared by ultrapure water machine；Nitrogen (>99.999%).
2, pre-treatment
2.1 Standard Stock solutions
10mg (being accurate to 0.1mg) citrus red2s number and Sudan red dyes (including I, II, III, IV type) standard are weighed respectively
Substance dissolves in volumetric flask, with acetonitrile and is settled to 50mL, is configured to citrus red2 number and the Sudan that concentration is about 200 μ g/mL
The Standard Stock solutions of red dye (including I, II, III, IV type), in 4 DEG C of preservations.
2.2 standard working solution
A certain amount of above-mentioned 2.1 Standard Stock solutions are drawn respectively, and it is respectively 100ng/mL, 200ng/ to be diluted to concentration step by step
The series standard working solution of mL, 1 μ g/mL, 2 μ g/mL, 4 μ g/mL.
When ultra performance liquid chromatography-tandem mass spectrometer is detected, due to the influence of matrix effect, lead to target compound
Ion enhancing or inhibiting effect occurs, therefore quantitative using bare substrate mark-on standard curve.Known vehicle solution is taken to add
Enter certain density standard working solution, it is 5 μ g/kg, 10 μ g/kg, 50 μ g/kg, 100 μ g/ to obtain 5 kinds of illegal dye contents
The blank mark-on sample of kg, 200 μ g/kg.
2.3 sample
It weighs 2g samples (being accurate to 0.01g) and 10mL acetonitriles is added in 50mL centrifuge tubes, 2g chlorine is added in ultrasonic 20min
Change sodium, acutely shake 1min, ultrasonic 10min, 3min is centrifuged with 8000r/min rotating speeds, is taken out in acetonitrile layer to 15mL centrifuge tubes,
It dries up under 40 DEG C of nitrogen streams, is dissolved with 3mL acetonitrile-dichloromethanes (1+99) solution, it is to be clean.
Liquid to be clean is transferred into column when liquid level is down to column bed with 6mL acetonitrile-dichloromethanes (1+99) activation pillar
Son is collected simultaneously scavenging solution.Twice with 3mL acetonitrile-dichloromethanes (1+99) rinse 15mL centrifuge tubes, continue collection and purification liquid.
It is concentrated to dryness under 40 DEG C of nitrogen streams, accurate 10mL formic acid-acetonitrile (1%) solution of drawing redissolves, and after crossing miillpore filter, waits for that machine is surveyed
It is fixed.
Above-mentioned sample includes blank mark-on sample and food substrate to be measured.
3, liquid chromatogram and Mass Spectrometry Conditions
3.1 liquid chromatogram parameters：
Liquid-phase chromatographic column：C18 (column length 75mm, column internal diameter 2.1mm；1.7 μm of filler particle size)；Flow velocity：0.30ml/min；Column
Temperature：40℃；Sampling volume：2.0μL；Mobile phase：Acetonitrile (A) and 0.1% aqueous formic acid (B)；Gradient elution program (A)：0-
3min:10% → 50%；3.0-5.0min:50% → 95%；5.0-7.0min:95% keeps 2min；7.0-7.1min:95%
→ 10%；7.1-9.0min:10%.
3.2 mass spectrometry parameters：
Capillary voltage:3.0KV；Orifice potential:25V；500 DEG C of desolvation temperature；Ion source temperature:150℃；Precipitation
Agent gas velocity:850L/Hr；Taper hole gas velocity:150L/Hr；Collision gas flow velocity:0.12mL/min；Scan mode:Multiple-reaction monitoring
(MRM)；Qualitative, quantitative ion pair, cracking voltage, residence time and the collision energy of citrus red2 number are shown in Table 2.
The mass spectrum reference conditions (ESI+) of citrus red2 No. 2 of table and tonyred (including I, II, III, IV type)
4, extraction standard solution-mass chromatogram and equation of linear regression
According to the condition of step 3 extraction standard solution is obtained after blank mark-on sample is carried out pre-treatment；By matrix mark
Quasi- solution distinguishes sample introduction according to concentration gradient, is detected with ultra performance liquid chromatography-tandem mass spectrum combined instrument, obtains matrix mark
Quasi- solution-mass chromatogram；Using the extraction standard chromatographic peak area of standard quality chromatogram as ordinate, with extraction standard solution
In each dyestuff respective concentration value be abscissa mapping, obtain standard curve and corresponding equation of linear regression.As shown in figure 3, being
Extraction standard containing a concentration of 25 μ g/kg citrus red2s number and a concentration of 25 μ g/kg tonyreds (including I, II, III, IV type) is molten
Liquid-mass chromatogram.
Using the extraction standard chromatographic peak area y of quota ion pair in extraction standard solution-mass chromatogram as ordinate, with
Concentration value (ng/mL) x of the corresponding ion of extraction standard solution is abscissa, makees standard curve, obtains citrus red2 number, tonyred
The equation of linear regression (as shown in table 3) of (including I, II, III, IV type).By table 3 as it can be seen that within the scope of 5-200 μ g/kg, citrus
The concentration and peak area of red No. 2 and Sudan red dyes (including I, II, III, IV type) are in good linear relationship, coefficient R2
More than 0.998；Citrus red2 number and tonyred (including I, II, III, IV type) response should be linear in standard curve in sample to be tested
In range, analyzed again after should then being diluted more than the range of linearity.
3 standard curve of table
5, the method rate of recovery, precision and detection limit
The rate of recovery (the rate of recovery=measured value/addition * of citrus red2 number and tonyred (including I, II, III, IV type)
100%), the results are shown in Table 4.By table 4 as it can be seen that citrus red2 number and Sudan red~IV the rate of recovery in 80.7%~114% model
In enclosing；
To various sample replication 6 times, measurement result all has good reproducibility, citrus red2 number and tonyred (packet
Include I, II, III, IV type) RSD be respectively less than 8.6%.
Using the method for adding target compound in known vehicle solution, the detection limit (S/ of citrus red2 number is measured
N>3) it is 5 μ g/kg, lower limit of quantitation (S/N>10) it is 10 μ g/kg；Detection limit (the S/N of tonyred (including I, II, III, IV type)>
3) it is 5 μ g/kg, lower limit of quantitation (S/N>10) it is 10 μ g/kg.
4 recovery testu of table
6. sample to be tested detects
(1) qualitative analysis
Sample is measured according to the condition of step 3 with ultra performance liquid chromatography-tandem mass spectrometer, obtains sample to be tested-matter
Chromatogram is measured, by the retention time of sample chromatographic peak compared with the retention time of reference colour spectral peak, and sample chromatographic peak
Relative abundance ratio determines the type of contained dyestuff in sample compared with reference colour spectral peak；Wherein, the reference colour spectral peak is by institute
It states standard working solution to be detected with ultra performance liquid chromatography-tandem mass spectrum combined instrument, obtained standard items-mass chromatogram
In obtain.As shown in Figure 1, citrus red2 number (10ng/mL) and tonyred (including I, II, III, IV type) (10ng/mL) standard items-
Mass chromatogram.
If there is chromatographic peak corresponding with the chromatographic peak in standard items-mass chromatogram in sample to be tested-mass chromatogram；
Then show to contain citrus red2 number or/tonyred (including I, II, III, IV type) in sample.Chromatographic peak refers to accordingly：Sample color
The retention time of spectral peak is compared with the retention time of reference colour spectral peak, and variation range is within ± 2.5%；And sample chromatographic peak
Relative abundance ratio compared with reference colour spectral peak, deviation be no more than table 5 as defined in range.Reference colour spectral peak refers to standard items-matter
Measure the chromatographic peak of the object in chromatogram.
With respect to the maximum allowable offset of abundance of ions when table 5 is qualitative
(2) quantitative to calculate
Using quantified by external standard method：According to the area of quota ion chromatographic peak in sample mass chromatogram, using returning for step 4
Return equation, the concentration of citrus red2 number/tonyred (including I, II, III, IV type) is calculated.
Invention described above embodiment is not intended to limit the scope of the present invention..
Those skilled in the art will readily occur to its of the present invention after considering specification and putting into practice the disclosure invented here
Its embodiment.The present invention is directed to cover the present invention any variations, uses, or adaptations, these modifications, purposes or
Person's adaptive change follows the general principle of the present invention and includes undocumented common knowledge in the art of the invention
Or conventional techniques.The description and examples are only to be considered as illustrative, and true scope and spirit of the invention are by following
Claim is pointed out.
It should be understood that the invention is not limited in the precision architectures for being described above and being shown in the accompanying drawings, and
And various modifications and changes may be made without departing from the scope thereof.The scope of the present invention is limited only by the attached claims.
Claims (6)
1. the universal test method of citrus red2 number and Sudan red dyes in a kind of food, which is characterized in that include the following steps：
(1) it prepares extraction standard solution and carries out high performance liquid chromatography-tandem mass detection
The Standard Stock solutions of the dyestuff of required test are diluted to the standard working solution of various concentration gradient, several pieces
Know and standard working solution is added in vehicle solution, obtains the blank mark-on sample of various concentration gradient；
(2) extraction standard solution-mass chromatogram and equation of linear regression are made
After blank mark-on sample is carried out pre-treatment, extraction standard solution is obtained；By extraction standard solution according to concentration gradient point
Other sample introduction is detected with ultra performance liquid chromatography-tandem mass spectrum combined instrument, obtains extraction standard solution-mass chromatogram；With
The extraction standard chromatographic peak area of standard quality chromatogram is ordinate, with the respective concentration value of each dyestuff in extraction standard solution
It maps for abscissa, obtains standard curve and corresponding equation of linear regression；
(3) qualitative analysis of sample to be tested
By food substrate to be measured after pre-treatment, sample to be tested is obtained；With ultra performance liquid chromatography-tandem mass spectrum combined instrument inspection
Sample to be tested is surveyed, sample to be tested-mass chromatogram is obtained, passes through the reservation of the retention time and reference colour spectral peak of sample chromatographic peak
Time compares, and the relative abundance ratio of sample chromatographic peak determines the type of contained dyestuff in sample compared with reference colour spectral peak；
Wherein, the reference colour spectral peak is to examine the standard working solution with ultra performance liquid chromatography-tandem mass spectrum combined instrument
It surveys, is obtained in obtained standard items-mass chromatogram；
(4) the quantitative calculating of sample to be tested
Using quantified by external standard method：Examination is calculated using the equation of linear regression of step (2) according to the area of sample chromatographic peak
The content of dyestuff in sample；
Wherein, dyestuff includes citrus red2 number, Sudan red type, Sudan II type, red Ⅲ type and SudanⅣ type；Step
(2) pre-treatment and in (3) is that sample is first passed through acetonitrile-sodium chloride system to extract, and takes out the drying of acetonitrile layer nitrogen, uses second
Nitrile-dichloromethane solution dissolving is redissolved using amino Solid Phase Extraction column purification with formic acid-acetonitrile solution, after crossing film, for super
High performance liquid chromatography-tandem mass combined instrument is detected, and the sample includes blank mark-on sample and food substrate to be measured；Institute
State the food substrate that food substrate to be measured includes high grease and/or high-moisture.
2. universal test method according to claim 1, which is characterized in that in the pre-treatment, acetonitrile-sodium chloride system
In, the ratio of acetonitrile-sodium chloride system and sample is 10mL:2g.
3. universal test method according to claim 1, which is characterized in that the ultra performance liquid chromatography-tandem mass spectrum
The parameter of combined instrument is as follows：
Liquid chromatogram parameter：
Liquid-phase chromatographic column：C18 liquid-phase chromatographic columns, column length 75mm, column internal diameter 2.1mm, 1.7 μm of filler particle size；
Column temperature：40℃；
Sampling volume：2.0μL
Mobile phase and gradient elution program：0-3min：0.1% aqueous formic acid 90% → 50%, acetonitrile 10% → 50%；3.0-
5.0min：0.1% aqueous formic acid 50% → 5%, acetonitrile 50% → 95%；5.0-7.0min：0.1% aqueous formic acid 5%,
Acetonitrile 95% keeps 2min；7.0-7.1min：0.1% aqueous formic acid 5% → 90%, acetonitrile 95% → 10%；7.1-
9.0min：0.1% aqueous formic acid 90%, acetonitrile 10%；
Mass spectrometry parameters：
Capillary voltage：3.0KV；Orifice potential：25V；Desolvation temperature：500℃；Ion source temperature：150℃；Desolventizing
Gas velocity：850L/Hr；Taper hole gas velocity：150L/Hr；Collision gas flow velocity：0.12mL/min；Scan mode：Multiple-reaction monitoring
MRM。
4. universal test method according to claim 1, which is characterized in that the pre-treatment includes the following steps：
It weighing 2g samples and 10mL acetonitriles is added in 50mL centrifuge tubes, ultrasonic 20min is added 2g sodium chloride, acutely shakes 1min,
Ultrasonic 10min centrifuges 3min with 8000r/min rotating speeds, takes out in acetonitrile layer to 15mL centrifuge tubes, dried up under 40 DEG C of nitrogen streams,
It is dissolved with 3mL acetonitrile-dichloromethane solution, it is to be clean；
Liquid to be clean is transferred into ammonia when liquid level is down to column bed with 6mL acetonitrile-dichloromethanes activation amino solid-phase extraction column
Base solid-phase extraction column, is collected simultaneously scavenging solution；Twice with 3mL acetonitrile-dichloromethane rinse 15mL centrifuge tubes, continue collection and purification
Liquid；It is concentrated to dryness under 40 DEG C of nitrogen streams, accurate 10mL formic acid-acetonitrile solution of drawing redissolves, after crossing miillpore filter, for liquid phase color
Spectrum-tandem mass spectrometry measures.
5. according to claim 1-4 any one of them universal test methods, which is characterized in that the food substrate to be measured includes
Fruit, jam, preserved fruit, fruit drink, chilli oil, thick chilli sauce, chafing dish bottom flavorings or meat products.
6. according to claim 1-4 any one of them universal test methods, which is characterized in that blank mark-on sample is each dyestuff
Content is the blank mark-on sample of 5 μ g/kg, 10 μ g/kg, 50 μ g/kg, 100 μ g/kg, 200 μ g/kg.
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