CN107748210A - The method that HPLC DAD detect a variety of synthetic dyestuffs in wheaten food simultaneously - Google Patents
The method that HPLC DAD detect a variety of synthetic dyestuffs in wheaten food simultaneously Download PDFInfo
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Abstract
The present invention relates to synthetic dyestuff detection technique field, more particularly to a kind of method that HPLC DAD detect a variety of synthetic dyestuffs in wheaten food simultaneously.Comprise the following steps：A, standard working solution and sample preparation；B, sample liquid is extracted；C, step B resulting solutions are taken to carry out HPLC DAD analyses；D, the content of measured matter is calculated with standard curve.Whereby, the present invention simplifies pre-treatment step, it is not necessary to carry out SPE and concentration step again, shorten the pre-treatment time by HPLC DAD detection methods.Using 400 600nm wavelength detectings, the interference of most of food additives and natural pigment is avoided.The method of the present invention is accurate, quick, easy, quantitative result is reliable, disposably can effectively detect ten kinds of synthetic food colors, be especially suitable for handling multiple batches of sample simultaneously.
Description
Technical field
The present invention relates to synthetic dyestuff detection technique field, more particularly to a kind of HPLC-DAD detect simultaneously it is a variety of in wheaten food
The method of synthetic dyestuff.
Background technology
Synthetic dyestuff is more using the arene compound such as coal tar or benzene, naphthalene as raw material, is synthesized by a series of organic reactions,
Itself have no no nutritive value.Synthetic food color has the days such as bright in luster, cost is cheap, strong coloring force, property are relatively stablized
The advantages of less than right pigment, thus it is widely used in food processing process.But synthetic dyestuff contains hazardous substance more, excessively takes the photograph
Membership causes damage to health, and its safety issue is increasingly subject to pay attention to, and various countries have strict limitation to this.Therefore it is accurate
It is determined that contained synthetic dyestuff is food-safe significant in amount food, or foods supervision department provides technology branch
Hold and punishing justification.
At present, detecting the national standard method of synthetic dyestuff has liquid chromatography, liquid chromatography-mass spectrography/mass spectrography, microtrabeculae chromatogram
Method etc., but most methods are only capable of detecting a few pigment simultaneously, and pretreatment process is cumbersome, the rate of recovery is low, especially I
There has been no the national standard for detecting a variety of pigments simultaneously, similar approach occasionally has been reported that also more with the simple matrix progress such as beverage, candy for state
Analysis, for complex matrices multicomponent, detected value must be inquired into simultaneously.Moreover basic unit's food inspection lab assistant is few, sample type
More, detection project is complicated, and workload is big, therefore a kind of method that can detect a variety of synthetic dyestuffs simultaneously is to improve detection efficiency drop
The necessary choice of low testing cost.
In summary, prior art there will naturally be inconvenience and defect in actual use, and it is therefore necessary to improved.
The content of the invention
For it is above-mentioned the defects of, it is an object of the invention to provide a kind of HPLC-DAD simultaneously detect a variety of synthesis in wheaten food
The method of pigment, by HPLC-DAD detection methods, simplify pre-treatment step, it is not necessary to carry out SPE again and concentration walks
Suddenly, the pre-treatment time is substantially reduced.Detected, avoided using 400-600nm wavelength of most chaff interferences without response
The interference of most of food additives and natural pigment.
To achieve these goals, the present invention provides the side that a kind of HPLC-DAD detects a variety of synthetic dyestuffs in wheaten food simultaneously
Method, comprise the following steps：
A, standard working solution and sample preparation are prepared
The preparation of standard working solution：Each standard pigment of precision weighing is simultaneously settled to required concentration with water respectively；
Sample preparation：From the blank sample without synthetic dyestuff, equivalent adds each standard pigment and sample is made；
B, sample liquid is extracted
Weigh the sample after crushing and be put into centrifuge tube, add extract solution ultrasonic after being vortexed uniformly；Then protein precipitation is added
Agent, after mixing on centrifuge centrifugal treating；Then supernatant is extracted and through membrane filtration；
C, step B resulting solutions are taken to carry out HPLC-DAD analyses, analysis condition is as follows：
C18 chromatographic columns：5 μm, 250 × 4.6mm；
Mobile phase：Methanol and ammonium acetate solution；
Gradient elution；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Sample size：10μL；
Detection wavelength：400-600nm；
D, the content of measured matter is calculated with standard curve.
The step B sample liquids are extracted：The sample after 2.0g is crushed is weighed in 15mL centrifuge tubes, add 8mL ethanol-
Ultrasonic extraction 10min after ammonia solution is vortexed uniformly；Add potassium ferrocyanide solution and acetic acid zinc solution, after mixing on centrifuge with
5000r/min centrifuges 5min, then takes supernatant through 0.22 μm of membrane filtration.
Prepare the water used in the standard working solution, ammonium acetate solution, potassium ferrocyanide solution and acetic acid zinc solution
For pure water.
The potassium ferrocyanide solution concentration is 106g/L, and the concentration of the acetic acid zinc solution is 220g/L, the ethanol-
Ammonia solution is:1mL ammoniacal liquor, it is that 70% ethanol mixes with 100mL concentration.
The addition of the potassium ferrocyanide solution and acetic acid zinc solution is 1ml.
The concentration of the ammonium acetate solution is 10mmol/L, and the condition of gradient elution is as shown in the table：
The concentration of the standard working solution is respectively：1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL and
20.0μg/mL。
The detection limit of a variety of synthetic dyestuffs：0.4-0.6mg/kg；Coefficient correlation is more than 0.998；Range of linearity 1-
20mg/L；The rate of recovery：88.4%-96.1%；RSD：0.83%-5.24%.
The synthetic dyestuff is ten kinds, is respectively：Lemon yellow, newly red, amaranth, famille rose, sunset yellow, temptation are red, bright
Indigo plant, acid red, erythrosine, Acid Orange II.
It is an object of the invention to provide a kind of method that HPLC-DAD detects a variety of synthetic dyestuffs in wheaten food simultaneously, pass through
HPLC-DAD detection methods, simplify pre-treatment step, it is not necessary to SPE and concentration step are carried out again, before substantially reducing
Processing time.Detected using 400-600nm wavelength of most chaff interferences without response, avoid most of food additives
With the interference of natural pigment.It disposably can effectively detect ten kinds of synthetic food colors.As a result show, method of the invention is accurate
Really, quickly, easy, quantitative result is reliable, be especially suitable for handling multiple batches of sample simultaneously.
Brief description of the drawings
Fig. 1 is the mixed standard solution chromatogram of the present invention；
Fig. 2 is the lemon yellow spectrogram of the present invention；
Fig. 3 is the newly red spectrogram of the present invention；
Fig. 4 is the carmine spectrogram of the present invention；
Fig. 5 is the amaranth spectrogram of the present invention；
Fig. 6 is the sunset yellow spectrogram of the present invention；
Fig. 7 is the temptation feux rouges spectrogram of the present invention；
Fig. 8 is the light blue spectrogram of the present invention；
Fig. 9 is the acid red spectrogram of the present invention；
Figure 10 is the erythrosine spectrogram of the present invention；
Figure 11 is the Acid Orange II spectrogram of the present invention.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention provides a kind of method that HPLC-DAD detects a variety of synthetic dyestuffs in wheaten food simultaneously, comprises the following steps：
A, standard working solution and sample preparation
The preparation of standard reserving solution：Precision weighing each standard substance water constant volume, concentration are respectively 500 μ g/mL.
4 DEG C of refrigerators preserve.
The preparation of standard working solution：It is respectively 1.0 μ g/mL, 2.0 that storing solution is diluted into concentration as needed during use
μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL series of tasks solution.
Mixed standard solution：The standard working solution of above-mentioned each pigment is selected to be made after mixing with concentration and equivalent.Its is dense
Degree is respectively 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL.
Used in the present invention ten kinds of standard pigments：Lemon yellow (GBW (E) 100001a), amaranth (GBW (E)
100002a), sunset yellow (GBW (E) 100003a), carmine (GBW (E) 100004a), light blue (GBW (E) 100005a), temptation
Red (GBW (E) 100164), newly red (SB05-219-2008), Acid Orange II CDCT-C15735000, are ground by national standard material
Center offer is provided；
Acid red (Lot:10504), erythrosine (Lot:40303) provided by Dr.EhrenstorferGmbH.
Sample preparation：From the blank sample without synthetic dyestuff, add above-mentioned ten kinds of standard pigments and sample is made.Every kind of mark
Quasi- pigment addition is 100 μ g.The sample is also referred to as mark-on sample.
B, sample liquid is extracted
Weigh the above-mentioned sample after crushing and be put into centrifuge tube, add extract solution ultrasonic after being vortexed uniformly；Then protein is added
Precipitating reagent, after mixing on centrifuge centrifugal treating；Then supernatant is extracted and through membrane filtration；
The step B sample liquids are extracted：The sample after 2.0g is crushed is weighed in 15mL centrifuge tubes, add 8mL ethanol-
Ultrasonic extraction 10min after ammonia solution is vortexed uniformly；Add potassium ferrocyanide solution and each 1.0mL of acetic acid zinc solution, after mixing from
5min is centrifuged with 5000r/min in scheming, then takes supernatant through 0.22 μm of membrane filtration；
The concentration of ammonium acetate solution is 10mmol/L, and compound method is：0.77g ammonium acetates, which are dissolved in water, is settled to 1000mL
And ultrasonic 10min.
Potassium ferrocyanide solution concentration is 106g/L, and compound method is：Weigh 106g potassium ferrocyanides [K4Fe (CN) 6
3H2O] add water to 1000mL.
The concentration of acetic acid zinc solution is 220g/L, and compound method is：Weigh 220g zinc acetates [Zn (CH3COO) 22H2O]
It is dissolved in a small amount of water, adds 30mL glacial acetic acids, be diluted with water to 1000mL.
Preferably, the present invention prepares above-mentioned ten kinds of standard pigments, ammonium acetate solution, potassium ferrocyanide solution and acetic acid
Water used in zinc solution is pure water.
Preferably, centrifuge of the invention is TGL-20M refrigerated centrifuges, and low-temperature centrifugation component is not easily decomposed, and is ensured
The stabilization of constitutive property in solution.
C, step B resulting solutions are taken to carry out HPLC-DAD analyses, analysis condition is as follows：
C18 chromatographic columns：5 μm, 250 × 4.6mm；
Mobile phase：The ammonium acetate solution of methanol and concentration 10mmol/L；
Gradient elution；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Sample size：10μL；
Detection wavelength：400-600nm；
Gradient elution is shown in Table 1；
Detection wavelength detection program is shown in Table 2.
Referring to table 1, it is preferred that the present invention to ensure gradient elution process and during result, especially follow-on test, it is steady
It is fixed.Mobile phase (the 10mmol/L ammonium acetate solutions of 5% methanol+95%) 4min is introduced into before sample introduction, is designated as in table 1：
“-4”。
Referring to table 1, it is preferred that gradient elution of the invention is in tri- times of 0-5min, 5-10 and 15-15.5min
The change of section mobile phase concentration proportioning turns to linear continuous variation.
D, the content of measured matter is calculated with standard curve；
Next, preferred, the determination explanation for being related to parameter in continuous mode are analyzed the present invention：
1. the optimization of chromatographic condition
The determination of 1.1 mobile phases
Mobile phase includes aqueous phase and organic phase.Detection of the food additives in liquid phase is more using ammonium acetate solution as water
Phase.Experiment finds that the concentration of ammonium acetate each component when in the range of 10-20mmol/L has preferable separation, but in gradient
Under elution requirement, the ammonium acetate of high concentration and the mixed proportion of organic phase easily cause crystallization analysis in the case where fluctuation is larger
Go out, so as to reduce the service life of chromatographic column.Therefore, aqueous phase of the invention uses ammonium acetate, and concentration is set as 10mmol/L.Have
Machine mutually selects methanol.
The determination of 1.2 column temperatures
The separating effect of each component when column temperature is respectively 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C has been investigated in experiment.It was found that column temperature
It can efficiently separate within this range, but as temperature raises, appearance time slightly shortens.Each component when but column temperature is 30 DEG C
Peak shape it is sharp and separating degree is preferable, therefore it is 30 DEG C to select column temperature.
The determination of 1.3 Detection wavelengths
According to national standard method more using 254nm as Detection wavelength, but it is experimentally confirmed, at this wavelength the sound of light blue
Interference that is very low, while easily causing the preservatives such as sorbic acid should be worth.
In view of also there is the hair that quantity does not wait in surveyed each component in addition to having stronger absorption in ultraviolet region, in each group fractionated molecule
Color and auxochrome group make it equally have stronger absorbing wavelength in visible region.Therefore, by using DAD detectors to each component
Standard liquid carries out spectral scan between 210~600nm and obtains respective spectrogram (see Fig. 2-Figure 11).Further according to going out for each component
Peak order, it is found that optimal absorption wave-length coverage of each component in visible region has coincidence in part-time section, by more suboptimums
Change, determine Detection wavelength and detection program (being shown in Table 2).
1.4 chromatogram
Referring to Fig. 1, separated by HPLC, the detection of DAD detectors, optimize ten kinds of artificial synthesized colors after chromatographic condition as described above
Element is efficiently separated, and sample disturbs under 400-600nm wavelength without obvious impurity.
2. the optimization of sample processing conditions
The selection of 2.1 extract solutions
Deionized water, 1% ammoniacal liquor and ethanol-ammonia solution extraction sample are used respectively, find deionized water and 1% ammoniacal liquor to red
Moss is red and the recovery rate of Acid Orange II is relatively low, and ethanol-ammonia solution can be with effectively extracting all pigments while have certain sink
The effect of shallow lake albumen.
Preferably, ethanol of the invention-ammonia solution collocation method is:1mL ammoniacal liquor, it is 70% second to add 100mL concentration
In alcohol.
The optimization of 2.2 precipitating reagents
Precipitating reagent is potassium ferrocyanide solution and acetic acid zinc solution 1:1 is used cooperatively.The two respectively each from 0.5ml,
1.0ml, 1.5ml, 2.0ml carry out experiment discovery, and 1.0ml precipitating reagents can effectively remove albumen in sample.But precipitating reagent mistake
The rate of recovery of pigment can be made reduce more, therefore select 1.0ml, be i.e. each 1.0mL of potassium ferrocyanide solution and acetic acid zinc solution.
3. the range of linearity and test limit of standard curve
To the mixed standard solution in abovementioned steps A, sample introduction is analyzed under abovementioned steps C chromatographic condition.Each hybrid standard
The concentration of solution is respectively 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL and 20.0 μ g/mL, final five groups of surveys
Test result.Using solution concentration as abscissa, linear regression is carried out by ordinate of corresponding peak area.During using 3 times of signal to noise ratio
Corresponding concentration, calculates detection limit.Regression equation, coefficient correlation, detection limit and the range of linearity are shown in Table 3.
As shown in Table 3, coefficient correlation generally meets or exceeds 0.9985；Detection limit：0.4-0.6mg/kg；Linearly
Scope 1-20mg/L.
4. the rate of recovery and Precision Experiment
Abovementioned steps B requirement respectively weighs blank sample and each 3 parts of mark-on sample, and parallel sample introduction 6 times, testing result is shown in Table 4.
In table 4, mark-on amount implication：Every kind of pigment addition is 100 μ g in mark-on sample, adds 8ml extractions altogether in stepb
Liquid and 2ml precipitating reagents, therefore the mark-on amount calculated is 10 μ g/mL.
As shown in Table 4, rate of recovery 88.4%-96.1%；RSD is 0.83%-5.24%.
The gradient elution program of table 1
The Detection wavelength of table 2 and detection program
The directrix curve regression equation of table 3, coefficient correlation and test limit
The yield of table 4 and Precision Experiment result
By result above, the present invention simplifies pre-treatment step, it is not necessary to again by HPLC-DAD detection methods
SPE and concentration step are carried out, substantially reduces the pre-treatment time.Using 400- of most chaff interferences without response
600nm wavelength is detected, and avoids the interference of most of food additives and natural pigment.It disposably can effectively detect ten kinds
Synthetic food color.As a result show, method of the invention is accurate, quick, easy, quantitative result is reliable, is especially suitable for locating simultaneously
Manage multiple batches of sample.
The present invention is combined as embodiment with foregoing ten kinds of synthetic dyestuffs, demonstrates the detection method of the present invention.Other are a variety of
Synthetic dyestuff combines, and is calculated and also may be used using sample pre-treatments of the present invention, HPLC-DAD analyses detection and standard curve
To detect clearly and effectively.This is no longer going to repeat them.
In summary, a variety of synthetic dyestuffs in wheaten food are detected simultaneously it is an object of the invention to provide a kind of HPLC-DAD
Method, by HPLC-DAD detection methods, simplify pre-treatment step, it is not necessary to carry out SPE and concentration step again, greatly
Shorten the pre-treatment time greatly.Detected using 400-600nm wavelength of most chaff interferences without response, avoid major part
The interference of food additives and natural pigment.It disposably can effectively detect ten kinds of synthetic food colors.As a result show, the present invention
Method it is accurate, quick, easy, quantitative result is reliable, be especially suitable for handling multiple batches of sample simultaneously.
Certainly, the present invention can also have other various embodiments, ripe in the case of without departing substantially from spirit of the invention and its essence
Know those skilled in the art when can be made according to the present invention it is various it is corresponding change and deformation, but these corresponding change and become
Shape should all belong to the protection domain of appended claims of the invention.
Claims (10)
1. a kind of method that HPLC-DAD detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that comprise the following steps：
A, standard working solution and sample preparation
The preparation of standard working solution：Each standard pigment of precision weighing is simultaneously settled to required concentration with water respectively；
Sample preparation：From the blank sample without synthetic dyestuff, equivalent adds each standard pigment and sample is made；
B, sample liquid is extracted
Weigh the sample after crushing and be put into centrifuge tube, add extract solution ultrasonic after being vortexed uniformly；Then protein precipitant is added, is mixed
After even on centrifuge centrifugal treating；Then supernatant is extracted and through membrane filtration；
C, the step B resulting solutions are taken to carry out HPLC-DAD analyses, analysis condition is as follows：
C18 chromatographic columns：5 μm, 250 × 4.6mm；
Mobile phase：Methanol and ammonium acetate solution；
Gradient elution；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Sample size：10μL；
Detection wavelength：400-600nm；
D, the content of measured matter is calculated with standard curve.
2. the method that HPLC-DAD according to claim 1 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
The step B sample liquids are extracted：The sample after 2.0g is crushed is weighed in 15mL centrifuge tubes, adds 8mL ethanol-ammonia solution whirlpool
Ultrasonic extraction 10min after rotation uniformly；Add potassium ferrocyanide solution and acetic acid zinc solution, with 5000r/ on centrifuge after mixing
Min centrifuges 5min, then takes supernatant through 0.22 μm of membrane filtration.
3. the method that HPLC-DAD according to claim 2 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
It is pure water to prepare the water used in the standard working solution, ammonium acetate solution, potassium ferrocyanide solution and acetic acid zinc solution.
4. the method that HPLC-DAD according to claim 2 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
The potassium ferrocyanide solution concentration is 106g/L, and the concentration of the acetic acid zinc solution is 220g/L, the ethanol-ammonia solution
For:1mL ammoniacal liquor, it is that 70% ethanol mixes with 100mL concentration.
5. the method that HPLC-DAD according to claim 2 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
The addition of the potassium ferrocyanide solution and acetic acid zinc solution is 1ml.
6. the method that HPLC-DAD according to claim 1 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
The concentration of the ammonium acetate solution is 10mmol/L, and the condition of gradient elution is as shown in the table：
7. the method that HPLC-DAD according to claim 1 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
The concentration of the standard working solution is respectively：1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL and 20.0 μ g/mL.
8. the method that the HPLC-DAD according to claim 1~7 any one detects a variety of synthetic dyestuffs in wheaten food simultaneously,
Characterized in that, the detection limit of a variety of synthetic dyestuffs：0.4-0.6mg/kg；Coefficient correlation is more than 0.998；Range of linearity 1-
20mg/L；The rate of recovery：88.4%-96.1%；RSD：0.83%-5.24%.
9. the method that HPLC-DAD according to claim 8 detects a variety of synthetic dyestuffs in wheaten food simultaneously, it is characterised in that
The synthetic dyestuff is ten kinds.
10. the method that HPLC-DAD according to claim 9 detects a variety of synthetic dyestuffs in wheaten food simultaneously, its feature exist
In described is that ten kinds of synthetic dyestuffs are respectively：Lemon yellow, newly red, amaranth, famille rose, sunset yellow, the red, light blue of temptation, acidity
Red, erythrosine, Acid Orange II.
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| 胡立立等: "HPLC．DAD法测定粗粮馒头中6种合成色素", 《食品科学》 * |
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