CN115436512A - Liquid chromatography tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food - Google Patents
Liquid chromatography tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food Download PDFInfo
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 33
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- GMMAPXRGRVJYJY-UHFFFAOYSA-J tetrasodium 4-acetamido-5-hydroxy-6-[[7-sulfonato-4-[(4-sulfonatophenyl)diazenyl]naphthalen-1-yl]diazenyl]naphthalene-1,7-disulfonate Chemical compound [Na+].[Na+].[Na+].[Na+].OC1=C2C(NC(=O)C)=CC=C(S([O-])(=O)=O)C2=CC(S([O-])(=O)=O)=C1N=NC(C1=CC(=CC=C11)S([O-])(=O)=O)=CC=C1N=NC1=CC=C(S([O-])(=O)=O)C=C1 GMMAPXRGRVJYJY-UHFFFAOYSA-J 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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Abstract
The invention discloses a liquid chromatography tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food, which comprises the steps of extracting the food by an alkalized methanol solution, centrifuging and filtering the food, measuring the food by LC/MS/MS (liquid chromatography/mass spectrometry), and quantifying by adopting a negative sample matrix standard curve and an external standard method. The method of the invention has simple operation and strong qualitative and quantitative capability, and can be used as a reliable method for detecting the content of 4 pigments in solid candy and liquid beverage food.
Description
Technical Field
The invention relates to a liquid chromatography tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food, belonging to the technical field of instrument detection.
Background
Various pigments are usually added in the modern food processing industry to improve the color of food and increase appetite, the use of edible pigments in food processing is very common, natural pigments have the defects of high cost, poor coloring power, poor stability and the like, and synthetic pigments are bright in color, good in stability and low in price and are widely favored by food production plants.
The synthetic pigment has the characteristics of bright color, strong tinting strength, multiple hues and low cost, so the synthetic pigment is widely applied. The synthetic pigment is prepared by taking chemical products such as benzene, toluene, naphthalene and the like as raw materials through a series of organic reactions, and the synthetic pigment has certain toxicity or carcinogenicity due to the mixing of an intermediate product. Therefore, the variety, range and addition amount of the artificially synthesized pigment allowed to be used in food are strictly regulated in various countries, and GB/T2760-2007 hygienic Standard for food additive use clearly regulates the range and the amount of the edible pigment allowed to be added in China.
Since the types of synthetic pigments allowed by regulations and the range of foods are limited, some illegal food producers are driven by interests, and can use the synthetic pigments in an excessive range, even pigments and dyes which are harmful to human bodies and cannot be used in foods are used, so that phenomena of excessive addition, illegal addition and the like occur in the using process of the pigments, and excessive intake of some pigments can cause health hazards, so that the analytical determination of the pigments in the foods is particularly important.
Eosin B, phloxine, sodium fluorescein salt and fast green belong to forbidden pigments in China, are used for solid candy and liquid beverage foods by illegal merchants, and a rapid and accurate detection method capable of simultaneously detecting the four forbidden pigments does not exist at present.
Disclosure of Invention
The invention provides a liquid chromatography tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food, wherein the four forbidden pigments are eosin B, fluorescein pink, fluorescein sodium salt and fast green.
The invention relates to a liquid chromatography tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food, which has the main technical principle that: extracting the food by using an alkalized methanol solution, centrifuging, filtering, measuring by using LC/MS/MS, and quantifying by using a negative sample matrix standard curve and an external standard method.
Further, the method specifically comprises the following steps:
(I) sample pretreatment
Weighing 5g of sample, placing the sample in a 50mL centrifuge tube, adding an alkaline methanol extracting solution to a constant volume of 50mL, carrying out vortex mixing for 30s, carrying out oscillation extraction for 1.5h, centrifuging at 4 ℃ for 3min at 5000r/min, taking 1mL of supernatant, filtering through a 0.22-micron filter membrane, observing the color of the filter membrane, if adsorption exists, taking the filtrate after 3-5 times, and then placing the filtrate in a sample injection vial for LC/MS/MS analysis.
(II) LC-MS/MS detection
(1) The chromatographic conditions were as follows:
a) A chromatographic column: porshell 120, EC-C18,3.0mm × 100mm, filler particle size 2.7 μm;
b) Mobile phase: phase A: water (0.025% formic acid +2mM ammonium acetate), phase B: acetonitrile;
c) Flow rate: 0.4mL/min;
d) Column temperature: 35 ℃;
e) Sample injection amount: 5 μ L
f) The gradient elution procedure was as follows:
| time (min) | A phase [% ]] | B phase [% ]] |
| 0.00 | 80 | 20 |
| 0.50 | 80 | 20 |
| 1.00 | 70 | 30 |
| 4.00 | 50 | 50 |
| 5.00 | 5 | 95 |
| 7.00 | 5 | 95 |
| 8.00 | 80 | 20 |
(2) Conditions of Mass Spectrometry
a) An ion source: electrospray (AJS ESI) ion source;
b) Scanning mode: scanning negative ions;
c) The detection mode comprises the following steps: multiple Reaction Monitoring (MRM);
c) Spray voltage (Capillary): -3000V;
d) Ion source temperature (Gas Temp): 300 ℃;
e) Gas Flow velocity (Gas Flow): 7L/min;
f) Atomizing gas (Nebulizer): 40psi;
g) Sheath Gas temperature (Sheath Gas Temp): 350 ℃;
h) Sheath Gas Flow (Sheath Gas Flow): 11L/min;
i) Other mass spectral conditions:
(3) Qualitative determination
Determining the sample and the matrix standard working solution according to the conditions of the liquid chromatography-mass spectrum/mass spectrum, and if the retention time of the mass chromatographic peak of the sample is consistent with that of the standard substance and all selected ions should appear, carrying out positive confirmation on the selected ions according to the types and relative abundance ratios of the qualitatively selected ion pairs; when in qualitative operation, the allowable deviation of the relative abundance does not exceed the range specified in the following table, and the existence of the corresponding detected object in the sample can be judged;
(4) Quantitative determination
Automatically feeding 5 mu L of each of 6-point negative sample matrix standard working solutions in a programmed sample feeding mode; and establishing a linear equation between the response value of the standard working solution and the content of 4 pigments in the sample by using a chemical workstation, and processing mass spectrum data of the sample solution.
The specific configuration of the negative sample matrix is the same as the pretreatment of the sample in the step (I), and the specific steps are as follows: adding alkalized methanol extract into pigment-free candy or beverage, and performing the above analysis steps.
Further, the preparation method of the alkalized methanol extracting solution comprises the following steps: 100mL of methanol, 900mL of water and 200 μ L of ammonia water are weighed and mixed uniformly respectively.
At present, pigment detection methods are more, most of edible pigments have related detection reports, but the methods for detecting eosin B, phloxine, sodium fluorescein salt and fast green simultaneously have no report,
in a Chinese journal paper 'simultaneous determination of 12 synthetic pigments in food by solid-phase extraction-high performance liquid chromatography', 12 detection steps of pigments are reported, wherein the 12 synthetic pigments are lemon yellow, brilliant black, sunset yellow, allure red, fast green, ponceau 2R, fluorescein sodium, ponceau 3R, patent blue, golden yellow powder, fluorescent pink, rose bengal; three of the present invention are included therein.
The difference from the invention lies in that firstly, the pretreatment mode is different; secondly, the detection mode is different, no literature data and standard methods for detecting the four pigments exist at present, and the traditional liquid chromatography method is tried in the early stage of searching and creating the detection method for the four pigments by the applicant, but the chromatography method has more impurities and poor selectivity. Finally, the invention adopts a liquid chromatography-mass spectrometry method.
The method adopts the negative sample matrix as a standard curve, effectively eliminates the matrix effect encountered in mass spectrometry and ensures that the quantitative result is more accurate.
Has the advantages that:
the method has simple operation and strong qualitative and quantitative capability, and can be used as a reliable method for detecting the content of 4 pigments in solid candy and liquid beverage foods.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a graph of a selected ion flow (500. Mu.g/kg concentration) of eosin B, phloxine, sodium fluorescein salt and fast green 4-pigment standards, from top to bottom.
FIG. 2 is a linear plot (concentration 20-500. Mu.g/kg) A of eosin B, phloxine, sodium fluorescein salt and fast green 4 pigments; the figure shows, from top to bottom, a linear plot of eosin B, phloxine, sodium fluorescein salt and fast green.
Detailed Description
Example 1
1. Examples liquid chromatography tandem mass spectrometry methods of eosin B, phloxine, sodium fluorescein salt, and fast green are provided for candy and beverages as examples.
2. Principle of the detection method of the embodiment
Extracting candy and beverage with 10% alkalized methanol solution, centrifuging, filtering, LC/MS/MS measuring, and quantifying by adopting negative sample matrix standard curve and external standard method.
3. Reagents and materials
Except for other provisions, all the reagents are analytically pure, and the water is primary water specified in GB/T6682.
3.1 methanol: and (4) carrying out chromatographic purification.
3.2 Ammonia water: and 4, high-grade purity.
3.3 extracting solution: 100mL of methanol (3.1), 900mL of water, and 200. Mu.L of ammonia water (3.2) were measured and mixed.
3.4 eosin B standard substance: the purity is more than or equal to 99.6 percent, and the SIGMA-ALDRICH is obtained.
3.5 fluorescent pink standard substance: the purity is more than or equal to 90 percent, and the SIGMA-ALDRICH is adopted.
3.6 fluorescein sodium salt standard substance: purity is more than or equal to 98.5 percent and Fluka.
3.7 fast green standard substance: the purity is more than or equal to 85 percent, and the SIGMA-ALDRICH is adopted.
3.8 eosin B Standard stock solution (1.0 mg/mL): accurately weigh 0.0250g of eosin B standard (3.4) into a 25mL volumetric flask, dissolve with the extract (3.3) and bring to volume. After mixing, the mixture was transferred to a liquid storage bottle and stored at 4 ℃.
3.9 fluorescent peach Red Standard stock solution (1.0 mg/mL): accurately weighing 0.0250g of fluorescent pink standard substance (3.5) in a 25mL volumetric flask, dissolving with the extracting solution (3.3) and fixing the volume to the scale. After mixing, the mixture was transferred to a liquid storage bottle and stored at 4 ℃.
3.10 Standard stock solution of sodium fluorescein salt (1.0 mg/mL): accurately weighing 0.0250g of fluorescein sodium salt standard substance (3.6) in a 25mL volumetric flask, dissolving with the extracting solution (3.3) and fixing the volume to the scale. After mixing, the mixture was transferred to a liquid storage bottle and stored at 4 ℃.
3.11 fast green standard stock (1.0 mg/mL): accurately weighing 0.0250g of fast green standard substance (3.7) in a 25mL volumetric flask, dissolving with the extract (3.3) and fixing to the mark. After mixing, the mixture was transferred to a liquid storage bottle and stored at 4 ℃.
3.12 Mixed intermediate solution (10. Mu.g/mL): accurately transferring 500 mu L of eosin B standard stock solution (3.8), phloxine B standard stock solution (3.9), fluorescein sodium salt standard stock solution (3.10) and fast green standard stock solution (3.11) into a 50mL volumetric flask, and diluting with the extracting solution (3.3) to a constant volume. After mixing, the mixture was transferred to a liquid storage bottle and stored at 4 ℃.
3.13 preparation of Standard working curves for four pigment bases
3.13.1 20ng/mL: accurately transferring 2. Mu.L of the mixed intermediate solution (3.12) into a sample injection vial, adding 998. Mu.L of the negative sample matrix solution, and uniformly mixing.
3.13.2 50ng/mL: accurately transferring 5. Mu.L of the mixed intermediate solution (3.12) into a sample injection vial, adding 995. Mu.L of the negative sample matrix solution, and uniformly mixing.
3.13.3 80ng/mL: accurately transferring 8. Mu.L of the mixed intermediate solution (3.12) into a sample injection vial, adding 992. Mu.L of the negative sample matrix solution, and uniformly mixing.
3.13.4 100ng/mL: accurately transferring 10 μ L of the mixed intermediate solution (3.12) into a sample injection vial, adding 990 μ L of the negative sample matrix solution, and mixing.
3.13.5 200ng/mL: accurately transferring 20 mu L of mixed intermediate solution (3.12) into a sample injection vial, adding 980 mu L of negative sample matrix solution, and mixing uniformly.
3.13.6 500ng/mL: accurately transferring 50 mu L of mixed intermediate solution (3.12) into a sample injection vial, adding 950 mu L of negative sample matrix solution, and mixing uniformly.
4. Apparatus and device
4.1 weighing balance: sartorius BS423S, sensory was 0.001g.
4.2 liquid chromatography-tandem mass spectrometer: an ESI source is provided.
4.3, a mixer: MODEL M37610-26.
4.4 Oscillator.
4.5 freezing centrifuge: is more than or equal to 5000r/min.
4.6 Milli-Q high purity water preparation instrument.
4.7 Polypropylene centrifuge tubes: 50mL.
4.8 microfiltration membrane: 0.22 μm.
4.9 injector: 1mL.
5. Analytical procedure
Weighing 5g (accurate to 0.001 g) of sample in a 50mL centrifuge tube, adding the extracting solution to a constant volume of 50mL, vortex and mixing uniformly for 30s, extracting for 1.5h by oscillation, centrifuging for 3min at 4 ℃ at 5000r/min, taking 1mL of supernatant, filtering through a 0.22 mu m filter membrane (observing the color of the filter membrane, and taking the filtrate after 3-5 times if adsorption is available) in a sample introduction vial, and performing LC/MS/MS analysis.
6. Measurement of
6.1 chromatographic conditions
a) And (3) chromatographic column: porshell 120, EC-C18,3.0mm X100 mm, filler particle size 2.7 μm.
g) Mobile phase: phase A: water (0.025% formic acid +2mM ammonium acetate), phase B: and (3) acetonitrile.
h) Flow rate: 0.4mL/min
i) Column temperature: 35 deg.C
j) Sample introduction amount: 5 μ L
k) Gradient of gradient
| Time (min) | A phase [% ]] | B phase [% ]] |
| 0.00 | 80 | 20 |
| 0.50 | 80 | 20 |
| 1.00 | 70 | 30 |
| 4.00 | 50 | 50 |
| 5.00 | 5 | 95 |
| 7.00 | 5 | 95 |
| 8.00 | 80 | 20 |
6.2 Mass Spectrometry conditions
a) An ion source: electrospray (AJS ESI) ion source;
b) Scanning mode: scanning negative ions;
c) The detection mode is as follows: multiple Reaction Monitoring (MRM);
c) Spray voltage (Capillary): -3000V;
d) Ion source temperature (Gas Temp): 300 ℃;
e) Gas Flow velocity (Gas Flow): 7L/min;
f) Atomizing gas (Nebulizer): 40psi;
g) Sheath Gas temperature (Sheath Gas Temp): 350 ℃;
h) Sheath Gas Flow (Sheath Gas Flow): 11L/min;
i) Other Mass Spectrometry conditions
6.3 qualitative determination
And (3) determining the sample and the matrix standard working solution according to the conditions of the liquid chromatography-mass spectrum/mass spectrum, and if the retention time of the mass chromatographic peak of the sample is consistent with that of the standard substance and all selected ions should appear, carrying out positive confirmation on the selected ions according to the types and relative abundance ratios of the qualitatively selected ion pairs. When the relative abundance of the sample is qualitative, the allowable deviation does not exceed the range specified in the following table, and the corresponding detected object can be determined to be present in the sample.
6.4 quantitative determination
In the sample injection manner, 5 μ L of each of 6-point negative sample matrix standard working solutions (3.17) was automatically injected. And establishing a linear equation between the response value of the standard working solution and the content of 4 pigments in the sample by using a chemical workstation, and processing mass spectrum data of the sample solution.
The linear curves of 4 pigments, eosin B, phloxine, sodium fluorescein salt and fast green, are respectively:
eosin B: y =17.924887 x +48.480923 2 ＝0.99977279。
Fluorescent pink: y =11.934608 x-157.997751, R 2 ＝0.99511909。
Fluorescein sodium salt: y =241.920109 x-2413.302600, R 2 ＝0.99809118。
Fast green: y =0.664732 x-8.897029, R 2 ＝0.99716672。
6.5 calculation of results
Quantification by external standard method, calculated as follows:
in the formula:
x-the pigment content of the sample in micrograms per kilogram (. Mu.g/kg)
C mass concentration of pigment in sample solution to be measured, unit is microgram per milliliter (mu g/L)
m-weight of sample in grams (g)
V-Total volume of sample solution to be measured in milliliters (mL)
K-dilution factor of sample liquid
The calculation result retains three significant digits, and blank values are deducted from the final result.
Example 2 solvent blank experiment and sample addition experiment
A solvent blank experiment and a sample addition experiment were performed on the method provided in example 1.
2.1 solvent blank
The samples were not added and treated in the same manner as the samples.
2.2 sample addition experiment
5g of a sample to be tested was weighed, and 150, 250, and 500. Mu.L of the mixed extract was added thereto and treated in the same manner as the sample. And (5) quantifying according to a negative sample matrix standard curve, and calculating the recovery rate, wherein the recovery rate is between 80 and 110 percent.
The specific data are as follows:
the detection limit of the method is 200 mug/kg.
When the tandem mass spectrometer is used for analysis, matrix effect can generate certain influence on a quantitative result, so that the matrix standard of a negative sample is used for quantification.
Example 3 uncertainty evaluation
1.A class uncertainty evaluation
Performing A-type uncertainty pre-evaluation by using a quality control sample result (adding and recycling) as a data source; the concrete method is as follows:
under the condition of uniform sample matrix, making 6 times of addition recovery experiments with same addition level in parallel, and calculating standard deviation of result average value of n times (n is greater than or equal to 6)
The standard uncertainty, as a (random) variation of the total repeated experiment, has a relative uncertainty component u of
Taking fluorescent pink as an example, 6 addition recovery measurements (addition level of 300. Mu.g/kg) were performed on the same sample, and the results are shown in Table 1:
TABLE 1 measurement results
Component of its relative uncertainty
5363 evaluation of uncertainty of class 2.B
The laboratory is provided with a central air conditioner for controlling temperature all the year round, meanwhile, the laboratory is forbidden to open windows, and works in the same place all the year round, so the influence of the ambient temperature and the atmospheric pressure can be ignored, and therefore, in other uncertainty sources, only the components of possible non-linearities in the purity, the dilution process, the weighing process and the response of a measuring instrument of a reference substance can be considered.
The volumetric flask and the pipette used in the experimental process are calibrated to meet the A-grade or 1-grade standard; considering that the same volumetric flask and pipette gun cannot be used every time in actual work, the maximum capacity tolerance given by JJJG 646-2006 "pipette verification procedure" and JJG 196-2006 "conventional glass measuring device verification procedure" is used for the uncertainty evaluation.
2.1 purity deviation of Standard substance-induced uncertainty component u rel (c s )
The maximum deviation of the purity of the standard substance provided by the certificate of the standard substance used in the experiment is +/-0.5%, and the value thereof introduces an uncertainty component
2.2 uncertainty component u caused by the Standard substance weighing Process rel (m s )
Weighing standard substance with balance sensitivity of 0.0001g (number 200937LE 0058) and sample amount of 0.0250g; scale calibration certificate gives U rel =0.26mg (k = 2), the relative uncertainty component introduced by the standard substance weighing process
2.3 uncertainty component introduced during dilution of Standard substance
2.3.1 uncertainty component u introduced in constant volume process of standard stock solution rel (v 1 )
The volume indication tolerance of the 25mL single-marking volumetric flask used in the experiment is +/-0.03 mL, the flask obeys triangular distribution, and the standard uncertainty component caused by the volumetric flask is
Its relative uncertainty component
2.3.2 uncertainty component u introduced during preparation of Standard intermediate solution rel (v 2 )
The maximum capacity tolerance of the 1mL pipette used in the experiment at the 0.5mL assay point is + -1.0%, and the maximum capacity tolerance follows a triangular distribution, the value of which causes a relative uncertainty component of
The tolerance of the volume indication value of the 50mL single-marking volumetric flask is +/-0.10 mL, the three-dimensional distribution is obeyed, and the standard uncertainty component caused by the volumetric flask is
The component of relative uncertainty caused by its value is
Synthesizing the above uncertainties, then u rel (v 2 )＝0.41％。
2.3.3 uncertainty component u introduced during Standard working Curve formulation rel (v 3 )
In the experimental process, 20.0, 50.0, 80.0, 100.0, 200.0 and 500.0ng/mL standard working solutions are prepared.
Table 2: uncertainty introduced during preparation of standard solutions of different concentrations
Synthesizing the above uncertainties to obtain u rel (v 3 )＝7.69％。
2.4 uncertainty component u introduced during the weighing of the sample to be measured rel (m)
A balance (number 200837LE 0089) with the sensitivity of 0.001g is used for weighing a sample to be measured, and the sample weighing amount is 5g; scale calibration certificate gives U rel =0.74mg (k = 2), the sample weighing process introduces a relative uncertainty component of
2.5 constant volume introduction of the uncertainty component u of the sample rel (v 4 )
After being processed, a sample is subjected to constant volume to 50mL by using a 50mL pointed-bottom centrifuge tube; according to JJG 10-2005 Special glass measuring instrument calibration protocol, the maximum capacity allowable error of a 50mL pointed-bottom centrifuge tube at the capacity of 5-50mL is +/-1.0 mL, and the maximum capacity allowable error follows triangular distribution, and the value of the maximum capacity allowable error causes the standard uncertainty component to be
With a relative uncertainty component of
2.6 detecting the uncertainty component u caused by the instrument rel (e)
The instrument used in the detection is Agilent 1260/6460 liquid chromatography-mass spectrometer, and U is given by a calibration certificate after calibration rel =2% (k = 2); component of instrumental induced uncertainty
3. Calculating the uncertainty of the synthesis standard
The relative standard uncertainties in Table 3 are combined, then u rel ＝7.69％。
Table 3: relative standard uncertainty source summary table
4. Calculating an extension uncertainty
Selecting 95% of the inclusion interval, with an inclusion factor of 2, and an extension uncertainty of U rel ＝15.38％(k＝2)。
5. Eosin B, sodium fluorescein salt and fast Green uncertainty assessment
Eosin B, sodium fluorescein salt and fast green uncertainty evaluations reference the fluorescent pink uncertainty evaluation procedure.
Choosing 95% of the inclusion interval, with an inclusion factor of 2, then the eosin B spread uncertainty is U rel ＝15.98％(k＝2)。
Selecting 95% of the inclusion interval, the inclusion factor is 2, and the uncertainty of the expansion of the sodium fluorescein salt is U rel ＝16.08％(k＝2)。
Selecting 95% of the inclusion interval, with an inclusion factor of 2, a fast green extension uncertainty of U rel ＝15.70％(k＝2)。
Example 4 specific application of the detection method
By using the method described in example 1, 4 pigments are detected in a large-batch detection process for multiple batches of samples sampled from the candy beverages for import and export commodities and the candy beverages sold in the market in the business of the applicant's director, so that negative samples without the four pigments are effectively eliminated, and a plurality of positive samples are timely found. For example: a ginger candy contains sodium fluorescein salt, and spinach juice beverage contains a small amount of fast green. Makes contributions to the monitoring of food safety risks in domestic markets and the safety of imported and exported foods.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (3)
1.A liquid chromatography-tandem mass spectrometry method for simultaneously detecting 4 forbidden pigments in food is characterized in that the food is extracted by an alkalized methanol solution, centrifuged and filtered, then measured by LC/MS/MS, and quantified by adopting a negative sample matrix standard curve and an external standard method.
2. The liquid chromatography-tandem mass spectrometry method for simultaneously detecting 4 banned pigments in food according to claim 1, which specifically comprises the following steps:
(I) sample pretreatment
Weighing 5g of sample, placing the sample in a 50mL centrifuge tube, adding an alkaline methanol solution to a constant volume of 50mL, carrying out vortex mixing for 30s, carrying out oscillation extraction for 1.5h, centrifuging the sample at 4 ℃ for 3min at 5000r/min, taking 1mL of supernatant, filtering the supernatant with a 0.22 mu m filter membrane, observing the color of the filter membrane, if adsorption exists, taking the filtrate after 3-5 times, and then placing the filtrate in a sample injection vial for LC/MS/MS analysis;
(II) LC-MS/MS detection
(1) The chromatographic conditions were as follows:
a) A chromatographic column: porshell 120, EC-C18,3.0mm X100 mm, filler particle size 2.7 μm;
b) Mobile phase: phase A: water (0.025% formic acid +2mM ammonium acetate), phase B: acetonitrile;
c) Flow rate: 0.4mL/min;
d) Column temperature: 35 ℃;
e) Sample introduction amount: 5 μ L
f) The gradient elution procedure was as follows:
(2) Conditions of Mass Spectrometry
a) An ion source: electrospray (AJS ESI) ion source;
b) Scanning mode: scanning negative ions;
c) The detection mode is as follows: multiple Reaction Monitoring (MRM);
c) Spray voltage (Capillary): -3000V;
d) Ion source temperature (Gas Temp): 300 ℃;
e) Gas Flow velocity (Gas Flow): 7L/min;
f) Atomizing gas (Nebulizer): 40psi;
g) Sheath Gas temperature (Sheath Gas Temp): 350 ℃;
h) Sheath Gas Flow (Sheath Gas Flow): 11L/min;
i) Other Mass Spectrometry conditions
(3) Qualitative determination
Determining the sample and the matrix standard working solution according to the conditions of the liquid chromatography-mass spectrum/mass spectrum, and if the retention time of the mass chromatographic peak of the sample is consistent with that of the standard substance and all selected ions should appear, carrying out positive confirmation on the selected ions according to the types and relative abundance ratios of the qualitatively selected ion pairs; when in qualitative operation, the allowable deviation of the relative abundance does not exceed the range specified in the following table, and the existence of the corresponding detected object in the sample can be judged;
(4) Quantitative determination
Automatically feeding 5 mu L of each of 6-point negative sample matrix standard working solutions in a programmed sample feeding mode; and establishing a linear equation of the response value of the standard working solution and the content of 4 pigments in the sample by using a chemical workstation, and processing the mass spectrum data of the sample solution.
3. The liquid chromatography-tandem mass spectrometry method for simultaneously detecting 4 banned pigments in food according to claim 2, wherein the preparation method of the alkalized methanol extract solution comprises the following steps: 100mL of methanol, 900mL of water and 200 μ L of ammonia water are weighed and mixed uniformly respectively.
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