CN102636592A - Method for simultaneous determination of a plurality of synthetic pigments in hot pot flavorings - Google Patents
Method for simultaneous determination of a plurality of synthetic pigments in hot pot flavorings Download PDFInfo
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Abstract
The invention discloses a method for simultaneous determination of a plurality of synthetic pigments in hot pot flavorings. The method comprises extraction, concentration, purification and detection of new red, amaranth, carmine, allura red, erythrosine, sunset yellow, acid red G, acid scarlet GR, Rhodamine B, Para Red, sudan I, sudan II, sudan III, sudan IV, sudan orange G, sudan red 7B and a plurality of other synthetic pigments. The method for simultaneous determination of a plurality of synthetic pigments in hot pot flavorings provided by the invention has the advantages of simple steps and high accuracy and precision.
Description
Technical field
The invention belongs to the chemical analysis detection range, assay method when being specifically related in the chafing dish bottom flavorings multiple synthetic dyestuff.
Background technology
Chafing dish bottom flavorings is to boil a kind of solid compound seasoner that forms by tens kinds of material mixing such as butter, capsicum, Chinese prickly ash, green onion, ginger, garlic and spices.Color and luster is scarlet to be big characteristics of chafing dish, and its main colour former is from the natural colouring matters such as capsicum red pigment in the capsicum, but illegal businessman is also arranged in order to reduce cost, improve appearance, in manufacturing process, adds various synthetic food colors.Synthetic food color is a raw material with benzene and homolog thereof usually, makes through a series of organic reactions such as oversulfonate, nitrated, halogenation and azoizations.Toxicologic study finds that some secondary colour have chronic toxicity or carcinogenicity, each national capital its usable range of strict control and use amount.
In view of the harmfulness of synthetic dyestuff to human body, the food that use use kind, use amount and the permission of synthetic food colors countries in the world all has clearly regulation.The secondary colour that Japan allow to use have amaranth, algae is red, temptation is red, carmine, flame is red, rose-bengal, orange, lemon yellow, sunset yellow, do not move back green FCF or its pale rose colour, light blue, totally 11 kinds; The U.S. allow to use has that A Luola is red, light blue, erythrosine, tangerine No. 2, fast green, indigo, lithol rubin, sunset yellow, lemon yellow and orange B etc.; European Union allow to use has Indian yellow, lemon yellow, quinoline yellow, sunset yellow FCF, famille rose, azorubine, amaranth, ponceau 4R, erythrosine, red 2G, lure red AC, patent blue V, indigo, brilliant blue FCF, brilliant black BN, brown FK, brown HT, lithol red BK directions etc.By the end of the year 1998, the secondary colour that China's approval allows to use have looks for that dish is red, to look for the red aluminium of dish color lake, famille rose, Ponceau 4R aluminum lake, red medicine red; Bare red aluminium color lake, newly red, New red aluminum lake; Lemon yellow, tartrazine aluminum lake, sunset yellow, Sunset yellow aluminum lake, bright orchid; Bright blue aluminium color lake, indigo, indigo aluminium color lake, sodium copper chlorophyllin, B-trailing plants B Bu Su, titania, temptation recklessly are red; Acid red etc., totally 21 kinds.
Because complicated, the matrix serious interference of chafing dish bottom flavorings composition, it is big to extract difficulty by conventional method when detecting pigment, and extracting is incomplete, influences the accuracy of measuring, and therefore is necessary to set up the efficient and accurate assay method of synthetic dyestuff in the chafing dish bottom flavorings.Set up easy, reliable, quick and sensitive analytical approach for the protection health,, guarantee food security, promote the health and the stable development of China's chafing dish industry, promote international trade, all have crucial meaning for the product quality of monitoring chafing dish bottom flavorings.
Summary of the invention
Assay method when the purpose of this invention is to provide in a kind of chafing dish bottom flavorings multiple synthetic dyestuff.
For realizing that the technical scheme that above-mentioned purpose the present invention adopts is:
Assay method multiple synthetic dyestuff time the in the chafing dish bottom flavorings, step is following:
1) extracts sample
Take by weighing the 5.00g sample to be tested in the 50mL centrifuge tube, add 20mL methyl alcohol-acetone soln, 60 ℃ of water-bath 10min, grease melt back vortex mixing, and the centrifugal 5min of 5000rpm is transferred to 150mL with supernatant and revolves in the steaming bottle; Add 20mL methyl alcohol-acetone soln, repeat aforesaid operations 1 time; Add 15mL 2mol/L urea methanol solution, repeat aforesaid operations 2 times; Merge each supernatant and steam in the bottle in revolving, 40 ℃ of underspins steam and are concentrated near doing.
2) isolation of purified sample
Use 5mL ethyl acetate-cyclohexane solution, 5mL water wash step 1 respectively) in revolve and steam bottle each 2 times, and cleansing solution is transferred in the 50mL centrifuge tube vortex mixing 1min, centrifugal 5 min of 5000rpm; Ethyl acetate in the centrifuge tube-cyclohexane layer is transferred in the 10mL color comparison tube, and 40 ℃ of following nitrogen are concentrated near doing, and with residue in ethyl acetate-cyclohexane solution dissolving color comparison tube and be settled to 5mL, must treat the fat-soluble sample of GPC purification; Add the 1mL phosphoric acid solution to the centrifuge tube aqueous phase, mixing must be treated the water-soluble sample that SPE purifies.
A, SPE purify
Water-soluble sample all is transferred in the polyamide chromatographic column, with 2～3mL/min chromatographic column of flowing through, simultaneously with 5mL 0.1% phosphoric acid solution washing centrifuge tube and be transferred in the chromatographic column; When treating that test solution has just arrived the chromatographic column top, in chromatographic column, add 5mL 80% methanol solution drip washing again and find time, use 10mL 5% ammoniacal liquor-methanol solution wash-out at last; 3mL eluent before discarding; Collect back 7mL eluent in the 10mL color comparison tube, and with methanol constant volume to scale, SPE purifies sample.
B, GPC purify
Fat-soluble sample is carried out GPC by following program purify, obtain GPC and purify sample;
Gel permeation chromatograph: decontaminating column 400 mm * 25 mm, interior dress Bio-Beads, S-X3,38 μ m～75 μ m fillers;
Moving phase: cyclohexane-ethyl acetate;
Flow velocity: 4.7 mL/min;
Sample size: 4.5 mL;
Beginning acquisition time: 13 min;
Finish acquisition time: 20 min.
3) concentrate SPE and purify sample and GPC purification sample
32.9mL GPC purification sample and 9.0mL SPE purification sample are transferred to same revolving in the steaming bottle; 40 ℃ of underspins steam and are concentrated near doing; Revolve with the washing of 5mL methyl alcohol-acetone soln and to steam bottle and be transferred in the scale test tube, concentrate near doing, add residue in 0.9mL methyl alcohol-acetone soln dissolving scale test tube at 40 ℃ of following nitrogen; After filtering membrane filters, supply HPLC to measure; Filtering membrane is 0.45 mm hydrophilic polyethersulfone membrane.
4) HPLC condition
Chromatographic column: C
18, 250 mm * 4.6 mm (internal diameter), 5 μ m;
Moving phase: methyl alcohol-0.01mol/L phosphate buffer, gradient elution;
Detect wavelength: program variable wavelength, 0～13.7min 520nm; 13.71～15min 420nm; 15.01～30min 520nm;
Column temperature: 30 ℃;
Flow velocity: 1.0mL/min;
Sample size: 10 μ L;
Back working time: 5.0min.
Obtain liquid chromatogram.
5) result calculates
With chromatographic data processor or by formula the content of synthetic dyestuff in the sample is calculated in (1), and result of calculation must the deduction blank value:
（1）
In the formula:
X
i--the content of test substance in the sample to be tested, unit are every kilogram of milligram;
c
i--the solution concentration of the test substance that obtains from liquid chromatogram, unit is every milliliter of a microgram;
V--the final constant volume of appearance liquid, unit are milliliter;
M--the sample to be tested quality of final samples liquid representative, unit is gram.
The elution program of gradient elution described in the step 4) is following:
Time/min methyl alcohol/% 0.01mol/L phosphate buffer/%
0 10 90
14 100 0
30 100 0。
Compare the present invention with existing detection method and can detect water-soluble and fat-soluble synthetic dyestuff in the multiple chafing dish bottom flavorings simultaneously, detect that step is easy, accuracy and precision is high.
Description of drawings
Fig. 1 is the liquid chromatogram (0.05 mg/L) of 16 kinds of synthetic dyestuff standard solution;
Horizontal ordinate is represented each pigment retention time among the figure, and ordinate is represented the response intensity (peak height) of each pigment; The the 5th and the 6th peak is respectively 9.742 and 9.923.
Embodiment
Below in conjunction with embodiment, specific embodiments of the invention is done further description.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
What present embodiment adopted is the bottom flavorings of spicy chaffy dish that Chongqing cygnet group produces, and specification is the 300g/ bag.Methyl alcohol, ethyl acetate and cyclohexane are selected the chromatographically pure level for use.
Concrete mensuration process is following:
1) extracts
Accurately take by weighing 5.00 g samples (being accurate to 0.01 g) in 50 mL centrifuge tubes, add respectively 20 mL methyl alcohol-acetone solns (1+1,
V/V), place 60 ℃ of water-bath 10 min, let grease melt after, vortex mixing 2 min, centrifugal 5 min of 5000 rpm are transferred to 150 mL with supernatant and revolve and steam in the bottle; Use again 20 mL methyl alcohol-acetone solns (1+1,
V/V) repeat aforesaid operations 1 time; Add 15 mL, 2 mol/L urea methanol solutions (containing 5% ammonia spirit) and repeat aforesaid operations 2 times; Merging each supernatant contracts near dried in 40 ℃ of underspin inspissations.
2) purify
Use respectively 5 mL ethyl acetate-cyclohexane solutions (1+1,
V/V), 5 mL water wash step 1) in revolve steam bottle each twice, and cleansing solution is transferred in the 50 mL centrifuge tubes vortex mixing 1 min, centrifugal 5 min of 5000 rpm.Ethyl acetate-cyclohexane layer is transferred in the 10 mL color comparison tubes, and 40 ℃ of following nitrogen concentrate near doing, with ethyl acetate-cyclohexane solution (1+1,
V/V) dissolved residue and be settled to 5 mL, treat that GPC purifies; To aqueous phase add 1 mL phosphoric acid solution (1+1,
V/V), mixing treats that SPE purifies.
A, SPE purify
Acidified water all is transferred in the polyamide chromatographic column; With 2～3 mL/min chromatographic column of flowing through; Simultaneously with 5 mL, 0.1% phosphoric acid solution washing centrifuge tube and be transferred in the chromatographic column; When treating that test solution has just arrived the chromatographic column top, in chromatographic column, add 5 mL, 80% methyl alcohol again and (contain 0.1% H
3PO
4) solution drip washing and finding time, use 10 mL, 5% ammoniacal liquor-methanol solution wash-out at last, discard preceding 3 mL eluents, collect back 7 mL eluents in 10 mL color comparison tubes, and with methanol constant volume to scale (10mL).
B, GPC purify
Gel permeation chromatograph: decontaminating column 400 mm * 25 mm (i.d.), interior dress Bio-Beads, S-X3,38 μ m～75 μ m fillers.
Moving phase: cyclohexane-ethyl acetate (1+1,
V/V);
Flow velocity: 4.7 mL/min;
Sample size: 4.5 mL;
Beginning acquisition time: 13 min;
Finish acquisition time: 20 min.
3) concentrate
32.9mL GPC eluent and 9.0 mL SPE eluents are transferred to revolve steam in the bottle, 40 ℃ of underspins steam and are concentrated near doing, with 5 mL methyl alcohol-acetone solns (1+1,
V/V) repeatedly washing is revolved and is steamed bottle and be transferred in the scale test tube on a small quantity, is concentrated near doing at 40 ℃ of following nitrogen, add 0.9mL methyl alcohol-acetone soln (1+1,
V/V) dissolved residue, behind the filtering membrane (0.45 mm hydrophilic polyethersulfone membrane), supply HPLC to measure.
4) liquid chromatography (HPLC) condition
Chromatographic column: C
18, 250 mm * 4.6 mm (internal diameter), 5 μ m, or suitable person;
Moving phase: methyl alcohol-0.01mol/L phosphate buffer (4.17), the gradient elution program is following:
Time/min methyl alcohol/% 0.01mol/L phosphate buffer/%
0 10 90
14 100 0
30 100 0；
Detect wavelength: program variable wavelength, 0～13.7min 520nm; 13.71～15min 420nm; 15.01～30min 520nm;
Column temperature: 30 ℃;
Flow velocity: 1.0 mL/min;
Sample size: 10 μ L;
Back working time: 5.0 min.
5) liquid chromatogram measuring
The content of middle measured object is selected the close standard operation solution of peak area per sample.The response of various synthetic dyestuffs all should be in the range of linearity of instrument in standard operation solution and the appearance liquid.Standard operation solution is measured with the interspersed sample introduction of appearance liquid equal-volume, and under above-mentioned chromatographic condition, the reference retention time of each target compound is seen middle table 1.The liquid chromatogram of standard solution is referring to Fig. 1.
The reference retention time of 16 kinds of synthetic dyestuffs of table 1
6) result calculates
With chromatographic data processor or by formula the content of synthetic dyestuff in the sample is calculated in (1), and result of calculation must the deduction blank value:
（1）
In the formula:
X
i--the content of test substance in the sample to be tested, unit are every kilogram (mg/kg) of milligram;
c
i--the solution concentration of the test substance that obtains from liquid chromatogram, unit is every milliliter of microgram (μ g/mL);
V--the final constant volume of appearance liquid, unit are milliliter (mL);
M--the sample to be tested quality of final samples liquid representative, unit is gram (g).
The concentration level that chafing dish bottom flavorings adds 10.0 mg/kg adds pigment to be measured and carries out recovery test, and average recovery rate and relative standard deviation that replicate determination is 6 times are seen table 2.
Table 2 mark-on average recovery rate and relative standard deviation
Can be found out that by above embodiment the embodiment of the invention detects multiple synthetic dyestuff in the chafing dish bottom flavorings through dissolving extraction, SPE column purification, GPC purification etc., method has higher sensitivity, accuracy and precision.
Claims (4)
1. assay method multiple synthetic dyestuff time the in the chafing dish bottom flavorings, step is following:
1) extracts sample
Take by weighing the 5.00g sample to be tested in the 50mL centrifuge tube, add 20mL methyl alcohol-acetone soln, 60 ℃ of water-bath 10min, grease melt back vortex mixing, and the centrifugal 5min of 5000rpm is transferred to 150mL with supernatant and revolves in the steaming bottle; Add 20mL methyl alcohol-acetone soln, repeat aforesaid operations 1 time; Add 15mL 2mol/L urea methanol solution, repeat aforesaid operations 2 times; Merge each supernatant and steam in the bottle in revolving, 40 ℃ of underspins steam and are concentrated near doing;
2) isolation of purified sample
Use 5mL ethyl acetate-cyclohexane solution, 5mL water wash step 1 respectively) in revolve and steam bottle each 2 times, and cleansing solution is transferred in the 50mL centrifuge tube vortex mixing 1min, centrifugal 5 min of 5000rpm; Ethyl acetate in the centrifuge tube-cyclohexane layer is transferred in the 10mL color comparison tube, and 40 ℃ of following nitrogen are concentrated near doing, and with residue in ethyl acetate-cyclohexane solution dissolving color comparison tube and be settled to 5mL, must treat the fat-soluble sample of GPC purification; Add the 1mL phosphoric acid solution to the centrifuge tube aqueous phase, mixing must be treated the water-soluble sample that SPE purifies;
A, SPE purify
Water-soluble sample all is transferred in the polyamide chromatographic column, with 2～3mL/min chromatographic column of flowing through, simultaneously with 5mL 0.1% phosphoric acid solution washing centrifuge tube and be transferred in the chromatographic column; When treating that test solution has just arrived the chromatographic column top, in chromatographic column, add 5mL 80% methanol solution drip washing again and find time, use 10mL 5% ammoniacal liquor-methanol solution wash-out at last; 3mL eluent before discarding; Collect back 7mL eluent in the 10mL color comparison tube, and with methanol constant volume to scale, SPE purifies sample;
B, GPC purify
Fat-soluble sample is carried out GPC by following program purify, obtain GPC and purify sample;
Gel permeation chromatograph: decontaminating column 400 mm * 25 mm, interior dress Bio-Beads, S-X3,38 μ m～75 μ m fillers;
Moving phase: cyclohexane-ethyl acetate;
Flow velocity: 4.7 mL/min;
Sample size: 4.5 mL;
Beginning acquisition time: 13 min;
Finish acquisition time: 20 min;
3) concentrate SPE and purify sample and GPC purification sample
32.9mL GPC purification sample and 9.0mL SPE purification sample are transferred to same revolving in the steaming bottle; 40 ℃ of underspins steam and are concentrated near doing; Revolve with the washing of 5mL methyl alcohol-acetone soln and to steam bottle and be transferred in the scale test tube, concentrate near doing, add residue in 0.9mL methyl alcohol-acetone soln dissolving scale test tube at 40 ℃ of following nitrogen; After filtering membrane filters, supply HPLC to measure;
4) HPLC condition determination
Chromatographic column: C
18
Moving phase: methyl alcohol-0.01mol/L phosphate buffer, gradient elution;
Detect wavelength: program variable wavelength, 0～13.7min 520nm; 13.71～15min 420nm; 15.01～30min 520nm;
Column temperature: 30 ℃;
Flow velocity: 1.0mL/min;
Sample size: 10 μ L;
Back working time: 5.0min;
Obtain liquid chromatogram;
5) result calculates
With chromatographic data processor or by formula the content of synthetic dyestuff in the sample is calculated in (1), and result of calculation must the deduction blank value:
（1）
In the formula:
X
i--the content of test substance in the sample to be tested, unit are every kilogram of milligram;
c
i--the solution concentration of the test substance that obtains from liquid chromatogram, unit is every milliliter of a microgram;
V--the final constant volume of appearance liquid, unit are milliliter;
M--the sample to be tested quality of final samples liquid representative, unit is gram.
2. according to assay method multiple synthetic dyestuff time the in the said chafing dish bottom flavorings of claim 1, it is characterized in that: the elution program of gradient elution described in the step 4) is following:
Time/min methyl alcohol/% 0.01mol/L phosphate buffer/%
0 10 90
14 100 0
30 100 0。
3. according to assay method multiple synthetic dyestuff time the in the said chafing dish bottom flavorings of claim 1, it is characterized in that: filtering membrane described in the step 3) is 0.45 mm hydrophilic polyethersulfone membrane.
4. according to assay method multiple synthetic dyestuff time the in the said chafing dish bottom flavorings of claim 1, it is characterized in that: the specification of chromatographic column described in the step 4) is 250 mm * 4.6 mm, 5 μ m.
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| CN102944639A (en) * | 2012-12-12 | 2013-02-27 | 华宝食用香精香料（上海）有限公司 | Distinguishing method for Sudan dyes in red pepper and tomatoes |
| CN103091314A (en) * | 2013-01-16 | 2013-05-08 | 深圳市计量质量检测研究院 | Rapid detection method for water-soluble azo dyes in foods |
| CN104655752A (en) * | 2015-02-10 | 2015-05-27 | 广西中烟工业有限责任公司 | Method for detecting acid brilliant scarlet GR in paper packaging material for foods |
| CN104820044A (en) * | 2015-05-05 | 2015-08-05 | 梧州市产品质量检验所 | Assay method of synthetic colorant in foods |
| CN105353025A (en) * | 2015-06-19 | 2016-02-24 | 南京工业大学 | Method for rapid determination of rhodamine B in food |
| CN105699465A (en) * | 2016-03-09 | 2016-06-22 | 福州大学 | Method for continuously separating synthetic pigments in multiple modes with pCEC (pressurized capillary electrochromatography) |
| CN108303481A (en) * | 2018-01-31 | 2018-07-20 | 山东省食品药品检验研究院 | The universal test method of citrus red2 number and Sudan red dyes in food |
| CN109425676A (en) * | 2017-08-26 | 2019-03-05 | 刘琳琳 | A kind of rapid detection method of Detection of Magdala in Food Through |
| CN109738541A (en) * | 2019-01-29 | 2019-05-10 | 江苏康达检测技术股份有限公司 | A kind of measuring method of Detection of Magdala in Food Through dyestuff |
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| CN110988201A (en) * | 2019-12-31 | 2020-04-10 | 欧陆分析技术服务(苏州)有限公司 | Method for efficiently detecting synthetic colorant |
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| CN102944639A (en) * | 2012-12-12 | 2013-02-27 | 华宝食用香精香料（上海）有限公司 | Distinguishing method for Sudan dyes in red pepper and tomatoes |
| CN103091314A (en) * | 2013-01-16 | 2013-05-08 | 深圳市计量质量检测研究院 | Rapid detection method for water-soluble azo dyes in foods |
| CN104655752B (en) * | 2015-02-10 | 2018-07-03 | 广西中烟工业有限责任公司 | The assay method of Acid Brilliant Scarlet GR in a kind of paper food packaging materials |
| CN104655752A (en) * | 2015-02-10 | 2015-05-27 | 广西中烟工业有限责任公司 | Method for detecting acid brilliant scarlet GR in paper packaging material for foods |
| CN104820044A (en) * | 2015-05-05 | 2015-08-05 | 梧州市产品质量检验所 | Assay method of synthetic colorant in foods |
| CN105353025A (en) * | 2015-06-19 | 2016-02-24 | 南京工业大学 | Method for rapid determination of rhodamine B in food |
| CN105699465A (en) * | 2016-03-09 | 2016-06-22 | 福州大学 | Method for continuously separating synthetic pigments in multiple modes with pCEC (pressurized capillary electrochromatography) |
| CN105699465B (en) * | 2016-03-09 | 2018-08-17 | 福州大学 | A kind of method that pressurization capillary electric chromatogram multi-mode continuously detaches synthetic dyestuff |
| CN109425676A (en) * | 2017-08-26 | 2019-03-05 | 刘琳琳 | A kind of rapid detection method of Detection of Magdala in Food Through |
| CN108303481A (en) * | 2018-01-31 | 2018-07-20 | 山东省食品药品检验研究院 | The universal test method of citrus red2 number and Sudan red dyes in food |
| CN109765325A (en) * | 2018-12-28 | 2019-05-17 | 沈阳出入境检验检疫局检验检疫综合技术中心 | A method of detection animal meat products neutral and alkali colorant |
| CN109738541A (en) * | 2019-01-29 | 2019-05-10 | 江苏康达检测技术股份有限公司 | A kind of measuring method of Detection of Magdala in Food Through dyestuff |
| CN110988201A (en) * | 2019-12-31 | 2020-04-10 | 欧陆分析技术服务(苏州)有限公司 | Method for efficiently detecting synthetic colorant |
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