CN111735886B - Pretreatment method of thermally processed food and detection method of acrylamide - Google Patents
Pretreatment method of thermally processed food and detection method of acrylamide Download PDFInfo
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a pretreatment method for thermally processed food and a detection method for acrylamide. The pretreatment method provided by the invention comprises the following steps: pulverizing the thermally processed food, adding 13 C 3 -mixing an acrylamide internal standard application liquid with water, extracting, mixing the obtained extraction system with dichloromethane for degreasing, purifying the sample extracting solution by adopting a GCB/PSA column, adding potassium bromide, a sulfuric acid solution and a potassium bromate solution, mixing, and deriving. According to the pretreatment method provided by the invention, through pure water extraction, dichloromethane degreasing, GCB/PSA column purification, acrylamide is generated into 2, 3-dibromoacrylamide after derivatization of potassium bromate and potassium bromide, the molecular weight is increased, the polarity is weakened, the chromatographic retention capacity is enhanced, the matrix interference phenomenon in LC-MS/MS detection is greatly reduced, and the detection accuracy is high.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a pretreatment method for thermally processed food and a detection method for acrylamide.
Background
Acrylamide is a well-known neurotoxin and quasi-carcinogen, and animal experiments and in vitro cell experiments prove that acrylamide can also cause genetic material to change. Thermally processed foods such as coffee and potato chips produce relatively high levels of acrylamide during the baking and frying process. The determination of acrylamide in food mainly comprises gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, for example, the existing standard detection method of acrylamide in food mainly comprises GB5009.204-2014 national standard for food safety-determination of acrylamide in food and SN/T2096-2008 detection method of acrylamide in food-gas chromatography-mass spectrometry isotope internal standard method. However, after the sample pretreatment provided by GB5009.204-2014 or SN/T2096-2008, the impurity interference of the sample is large, and the detection accuracy is not high.
Disclosure of Invention
The invention aims to provide a pretreatment method for thermally processed food and a detection method for acrylamide.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a pretreatment method of thermally processed food, which comprises the following steps:
mixing the hot processed food sample, 13 C 3 -mixing an acrylamide internal standard application liquid with water for extraction, mixing the obtained extraction system with dichloromethane for degreasing to obtain a sample extraction liquid;
purifying the sample extracting solution by adopting a GCB/PSA column to obtain a sample purifying solution;
and mixing the sample purifying solution, potassium bromide, a sulfuric acid solution and a potassium bromate solution, and deriving to obtain a sample solution to be detected.
Preferably, the extraction means comprises ultrasonic extraction or vortex extraction.
Preferably, the thermally processed food sample comprises a food product, coffee beans or a coffee beverage.
Preferably, the ratio of the mass of the thermally processed food sample to the volume of water is 1 g: (5-100) mL.
Preferably, the ratio between the mass of the thermally processed food sample and the volume of dichloromethane is 1 g: (5-25) mL.
Preferably, the molar ratio of the potassium bromate solution to the potassium bromide solution to the sulfuric acid solution is 1: (5-6): (3-4).
Preferably, the derivatization is carried out under sealed and light-tight conditions.
The invention provides a method for detecting acrylamide in thermally processed food, which comprises the following steps:
detecting the content of the 2, 3-dibromoacrylamide in the sample liquid to be detected obtained by the pretreatment method in the technical scheme by adopting a liquid chromatography-tandem mass spectrometry;
the amount of the 2, 3-dibromoacrylamide is calculated by acrylamide.
Preferably, the conditions of the liquid chromatography detection in the liquid chromatography tandem mass spectrometry detection include:
a chromatographic column: a UPLC BEH C18 column;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
mobile phase: the mobile phase A is a formic acid aqueous solution, and the volume percentage concentration of the formic acid aqueous solution is 0.05-0.3%; the mobile phase B is a methanoic acid solution, and the volume percentage concentration of the methanoic acid solution is 0.05-0.3%; the flow rate of the mobile phase is 0.2-0.6 mL/min;
and (3) an elution mode: gradient elution;
the procedure for the gradient elution was as follows:
0.00-3.00 min, wherein the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%;
3.00-5.00 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 85% at a constant speed, and the volume percentage of the mobile phase B is increased from 5% to 15% at a constant speed;
5.00-5.10 min, wherein the volume percentage of the mobile phase A is reduced from 85% to 0% at a constant speed, and the volume percentage of the mobile phase B is increased from 15% to 100% at a constant speed;
5.10-6.00 min, wherein the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is 100%;
6.00-6.10 min, wherein the volume percentage of the mobile phase A is increased from 0% to 95% at a constant speed, and the volume percentage of the mobile phase B is decreased from 100% to 5% at a constant speed;
and 6.10-7.50 min, wherein the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%.
Preferably, the conditions for mass spectrometry detection in the liquid chromatography tandem mass spectrometry detection include:
an ion source: an electrospray ion source;
ion source temperature: 150 ℃;
capillary voltage: 3.0 kV;
desolventizing temperature: 500 ℃;
the scanning mode is as follows: scanning positive ions;
the detection mode comprises the following steps: detecting multiple reactions;
atomizing: nitrogen gas or high purity nitrogen gas produced by a nitrogen generator;
back blowing of the taper hole: nitrogen gas or high purity nitrogen gas produced by a nitrogen generator;
collision gas: high purity argon gas.
The invention provides a pretreatment method of thermally processed food, which comprises the following steps: crushing the thermally processed food to obtain a thermally processed food sample; subjecting the thermally processed food sample to, 13 C 3 -mixing an acrylamide internal standard application liquid with water for extraction, mixing the obtained extraction system with dichloromethane for degreasing to obtain a sample extraction liquid; purifying the sample extracting solution by adopting a GCB/PSA column to obtain a sample purifying solution; and mixing the sample purifying solution, potassium bromide, a sulfuric acid solution and a potassium bromate solution, and deriving to obtain a sample solution to be detected. According to the pretreatment method provided by the invention, water is used as an extracting agent, the extraction rate of acrylamide is high, and the pretreatment method is safe and environment-friendly; fat can be extracted into a dichloromethane layer by dichloromethane degreasing, acrylamide is remained in an aqueous solution, the purpose of degreasing is realized, other substances except the acrylamide, such as acidic substances, pigments and the like, can be removed by GCB/PSA column purification, the purpose of further evolution is achieved, then the acrylamide is derived by potassium bromate and potassium bromide to generate 2, 3-dibromoacrylamide, the molecular weight is increased, the polarity is weakened, the chromatographic retention capacity is enhanced, the sample matrix interference is greatly reduced, and the accuracy of subsequent detection of the acrylamide by LC-MS/MS is improved. The detection method provided by the invention is simple, convenient and rapid, and has low detection limit and high accuracy. As shown in the results of examples, when the sample mass was 0.5g, the detection limit of acrylamide was 0.6. mu.g/kg, and the quantification limit was 2.0. mu.g/kg。
Drawings
FIG. 1 shows 2, 3-dibromoacrylamide and 13 C 3 -standard TIC profile of 2, 3-acrylamide;
FIG. 2 is a TIC map of a potato chip sample;
FIG. 3 is a TIC map of a coffee sample;
FIG. 4 is a standard spectrum of acrylamide and a TIC spectrum of a coffee sample detected according to GB 5009.204-2004.
Detailed Description
The invention provides a pretreatment method of thermally processed food, which comprises the following steps:
mixing the hot processed food sample, 13 C 3 -mixing an acrylamide internal standard application liquid with water for extraction, mixing the obtained extraction system with dichloromethane for degreasing to obtain a sample extraction liquid;
purifying the sample extracting solution by adopting a GCB/PSA column to obtain a sample purifying solution;
and mixing the sample purifying solution, the potassium bromide, the sulfuric acid solution and the potassium bromate solution, and deriving to obtain a sample solution to be detected.
In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
The invention adopts the steps of preparing a thermally processed food sample, 13 C 3 -mixing an acrylamide internal standard application liquid with water for extraction, mixing the obtained extraction system with dichloromethane for degreasing, and obtaining a sample extraction liquid.
In the present invention, the thermally processed food sample preferably comprises a food product, coffee beans or a coffee beverage. In the present invention, when the thermally processed food sample is a food product or coffee bean, the thermally processed food sample is preferably pulverized before use. The pulverization method of the present invention is not particularly limited, and pulverization methods known to those skilled in the art may be used. In the present invention, the particle size of the pulverized thermally processed food sample is preferably 50 to 120 mesh, and more preferably 80 mesh.
In the present invention, the 13 C 3 The concentration of the application liquid of the acrylamide internal standard is preferably 5.0. mu.g/mL; in the present invention, the quality of the thermally processed food sample and 13 C 3 the ratio of the volumes of the application liquids of the acrylamide internal standard is preferably 1 g: (40-400) mL. In the present invention, the 13 C 3 The amount of application liquid of the acrylamide internal standard is preferably chosen according to the type of the thermally processed food sample, the quality and the quality of the thermally processed food sample when the thermally processed food sample is a food product 13 C 3 -the ratio between the volumes of the application liquids of the acrylamide internal standard is preferably 1 g: (40-100) mL, more preferably 1 g: 80 mL; the food product, the thermally processed food sample, when the thermally processed food sample is coffee beans, the quality of the thermally processed food sample, and 13 C 3 the ratio of the volumes of the application liquids of the acrylamide internal standard is preferably 1 g: (200-400) mL, more preferably 1 g: 400 mL; the food product, the thermally processed food sample, when the thermally processed food sample is a coffee beverage, the food product, the thermally processed food sample, and the 13 C 3 The ratio of the volumes of the application liquids of the acrylamide internal standard is preferably 1 g: (20-100) mL, more preferably 1 g: 40 mL.
In the present invention, the ratio of the mass of the thermally processed food sample to the volume of water is preferably 1 g: (5-100) mL, more preferably 1 g: (9-100) mL. In the present invention, the amount of water is preferably selected according to the kind of the thermally processed food sample, and when the thermally processed food sample is a food product, the ratio of the mass of the thermally processed food sample to the volume of water is preferably 1 g: 20 mL; when the thermally processed food sample is coffee beans, the food product preferably has a mass to water volume ratio of 1 g: 100 mL; when the thermally processed food sample is a coffee beverage, the ratio of the mass of the food product to the volume of water in the thermally processed food sample is preferably 1 g: 9 mL.
In the present invention, the extraction means preferably comprises ultrasonic extraction or vortex extraction; the temperature of the extraction is preferably room temperature. In the invention, the power of the ultrasonic wave is preferably 400-500W, and more preferably 500W; the ultrasonic treatment time is preferably 5-30 min, and more preferably 5-10 min; in an embodiment of the present invention, the ultrasound is preferably performed in an ultrasonic cleaner (kunshan ultrasonic instruments ltd). In the invention, the vortex extraction time is preferably 4-6 min, and more preferably 5 min. In embodiments of the invention, the vortexing is preferably performed in a multi-sample vortex shaker (Heidolph group, germany).
In the present invention, the ratio of the mass of the thermally processed food sample to the volume of dichloromethane is preferably 1 g: (5-25) mL, more preferably 1 g: (10-15) mL. In the present invention, the amount of dichloromethane to be used is preferably selected according to the kind of the thermally processed food sample, and when the thermally processed food sample is a food product, the ratio of the mass of the thermally processed food sample to the volume of dichloromethane is preferably 1 g: 10 mL; when the thermally processed food sample is coffee beans, the ratio of the mass of the thermally processed food sample to the volume of methylene chloride is preferably 1 g: 25 mL; when the thermally processed food sample is a coffee beverage, the ratio of the mass of the thermally processed food sample to the volume of dichloromethane of the food product is preferably 1 g: 5 mL.
After the degreasing, the invention preferably further comprises performing vortex treatment and centrifugal separation on the degreased system, wherein the obtained supernatant is a sample extracting solution. In the invention, the time of the vortex treatment is preferably 4-6 min, and more preferably 5 min; in an embodiment of the invention, the vortexing process is preferably performed in a multi-sample vortex shaker (Heidolph group, germany); the purpose of the vortex treatment is to improve the extraction efficiency of acrylamide. In the invention, the speed of centrifugal separation is preferably 9000-11000 r/min, and more preferably 10000 r/min; the time for centrifugal separation is preferably 2-4 min, and more preferably 3 min.
After the sample extracting solution is obtained, the sample extracting solution is purified by adopting a GCB/PSA column to obtain the sample purifying solution.
In the present invention, the GCB/PSA column (graphitized carbon black/ethylene diamine-N-propyl column). In the invention, the flow rate of the sample extracting solution in the GCB/PSA column is preferably 1-3 mL/min, and more preferably 2 mL/min. In the present invention, the sample extraction liquid is preferably collected in a centrifuge tube. In the present invention, the GCB/PSA column is purged to remove acidic species, pigments, and impurities that affect the subsequent bromination reaction.
After obtaining the sample purifying solution, the invention mixes the sample purifying solution, potassium bromide, sulfuric acid solution and potassium bromate solution, and performs derivation to obtain the sample solution to be detected.
In the invention, the concentration of the potassium bromate solution is preferably 15-20 g/L, and more preferably 17 g/L. In the invention, the concentration of the sulfuric acid solution is preferably 0.2-0.4 mol/L, and more preferably 0.3 mol/L. In the present invention, the molar ratio of the potassium bromate solution, the potassium bromide solution and the sulfuric acid solution is preferably 1: (5-6): (3-4), more preferably 1:5: 3. In the present invention, the ratio of the volume of the sample purification solution to the mass of potassium bromide is preferably (5 to 15) mL: 0.75g, more preferably 10 mL: 0.75 g.
In the present invention, the derivatization is preferably carried out under sealed and light-tight conditions; the temperature of the derivatization is preferably room temperature; the derivatization time is preferably 25-35 min, and more preferably 30 min; the derivatization is preferably a standing derivatization. In the derivatization process, under the condition of sulfuric acid, potassium bromide reacts with potassium bromate to generate bromine water, then C ═ C double bonds on acrylamide and the bromine water generate addition reaction to generate 2, 3-dibromoacrylamide, the molecular weight of the 2, 3-dibromoacrylamide is large, the polarity is weakened, the chromatographic retention capacity is enhanced, and the matrix interference phenomenon in LC-MS/MS detection is greatly reduced.
After the derivatization, the method preferably further comprises the steps of adding a sodium thiosulfate solution into the derivatized system to remove residual bromine, adding ethyl acetate to perform vortex extraction, performing first centrifugal separation, drying the obtained supernatant, adding a formic acid aqueous solution to dissolve the supernatant, and performing second centrifugal separation to obtain the supernatant which is the sample liquid to be detected. In the invention, the concentration of the sodium thiosulfate solution is preferably 0.2-0.3 g/mL, and more preferably 0.25 g/mL; the using amount of the sodium thiosulfate solution is not specially limited, and the residual bromine in the system can be removed completely. In the present invention, the detection method of the residual bromine in the system is preferably visual, the bromine is yellow, and when the yellow color in the system is faded, the residual bromine is removed. In the invention, the volume ratio of the sample purifying solution to the ethyl acetate is preferably (5-15): (2-4), more preferably (5-15): 3. in the invention, the vortex extraction time is preferably 4-6 min, and more preferably 5 min; in embodiments of the invention, the vortexing is preferably performed in a multi-sample vortex shaker (Heidolph group, germany); the vortex extraction can remove the interference of fat on the matrix. In the invention, the speed of the first centrifugal separation and the speed of the second centrifugal separation are independently preferably 9000-11000 r/min, and more preferably 10000 r/min; the time is preferably 2 to 4min, and more preferably 3 min. In the invention, the blow-drying is preferably liquid nitrogen blow-drying; in the embodiment of the present invention, the liquid nitrogen blow-drying is preferably performed by using a nitrogen blower (Shanghai Anan spectral laboratory science and technology Co., Ltd.). In the invention, the volume percentage concentration of the formic acid aqueous solution is preferably 0.05-0.15%, and more preferably 0.1%; the volume ratio of the sample purifying solution to the formic acid aqueous solution is preferably (5-15): (0.5 to 1.5), more preferably (5 to 15): 1; the function of the formic acid aqueous solution is to dissolve out the 2, 3-dibromoacrylamide and the internal standard substance.
The invention provides a method for detecting acrylamide in thermally processed food, which comprises the following steps:
detecting the content of the 2, 3-dibromoacrylamide in the sample liquid to be detected obtained by the pretreatment method in the technical scheme by adopting liquid chromatography-tandem mass spectrometry (LC-MS/MS);
the amount of the 2, 3-dibromoacrylamide is calculated by acrylamide.
In the present invention, the liquid chromatography tandem mass spectrometry detection preferably comprises the following steps:
performing liquid chromatography tandem mass spectrometry detection on the sample liquid to be detected to obtain a sample chromatogram;
obtaining 2, 3-dibromoacrylamide according to the sample chromatogram map 13 C 3 -peak area of 2, 3-dibromoacrylamide; according to the relative peak area and the linear curve, adoptingCalculating the concentration of the 2, 3-dibromoacrylamide in the sample liquid to be detected by an internal standard method, and calculating the content of the 2, 3-dibromoacrylamide in the sample liquid to be detected according to the formula (1); the linear curve is a linear curve of the chromatographic relative peak area of the 2, 3-dibromoacrylamide and the concentration of the acrylamide; the amount of the 2, 3-dibromoacrylamide is calculated by acrylamide.
The invention carries out liquid chromatography tandem mass spectrometry detection on the sample liquid to be detected to obtain a sample chromatogram.
In the present invention, the conditions of the liquid chromatography detection in the liquid chromatography tandem mass spectrometry detection preferably include: the chromatographic column is preferably an UPLC BEH C18 column, and the specification of the UPLC BEH C18 column is preferably 1.7 μm and 2.1mm multiplied by 100 mm; the column temperature is preferably 30 ℃; the sample injection amount is preferably 10 mu L; the mobile phase is preferably a mobile phase A and a mobile phase B, the mobile phase A is preferably a formic acid aqueous solution, and the volume percentage concentration of the formic acid aqueous solution is preferably 0.05-0.3%, and more preferably 0.1-0.2%; the mobile phase B is preferably a methanoic acid solution, and the volume percentage concentration of the methanoic acid solution is preferably 0.05-0.3%, and more preferably 0.1-0.2%; the flow rate of the mobile phase is preferably 0.2-0.6 mL/min, more preferably 0.3-0.5 mL/min, and most preferably 0.3 mL/min; the detector is preferably a triple quadrupole mass spectrometer detector; the elution mode is preferably gradient elution. In the present invention, the volume percentages of the mobile phase a and the mobile phase B during the gradient elution are shown in table 1:
TABLE 1 liquid chromatography gradient elution conditions
| Time/min | Mobile phase A/volume% | Mobile phase B/volume% |
| 0.00 | 95 | 5 |
| 3.00 | 95 | 5 |
| 5.00 | 85 | 15 |
| 5.10 | 0 | 100 |
| 6.00 | 0 | 100 |
| 6.10 | 95 | 5 |
| 7.50 | 95 | 5 |
Namely, the procedure of the gradient elution is as follows:
0.00-3.00 min, wherein the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%;
3.00-5.00 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 85% at a constant speed, and the volume percentage of the mobile phase B is increased from 5% to 15% at a constant speed;
5.00-5.10 min, wherein the volume percentage of the mobile phase A is reduced from 85% to 0% at a constant speed, and the volume percentage of the mobile phase B is increased from 15% to 100% at a constant speed;
5.10-6.00 min, wherein the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is 100%;
the volume percentage of the mobile phase A is increased from 0% to 95% at a constant speed and the volume percentage of the mobile phase B is decreased from 100% to 5% at a constant speed for 6.00-6.10 min;
and 6.10-7.50 min, wherein the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%.
In the present invention, the conditions for mass spectrometry detection in the liquid chromatography tandem mass spectrometry detection preferably include: the ion source is preferably an electrospray ion source (ESI +); the ion source temperature is preferably 150 ℃; the capillary voltage is preferably 3.0 kV; the preferable desolvation temperature is 500 ℃; the scanning mode is preferably positive ion scanning; the detection mode is preferably multi-reaction detection (MRM); the atomizing gas is preferably nitrogen high-purity nitrogen generated by a nitrogen generator, and the purity of the high-purity nitrogen is preferably 99.999%; the taper hole back blowing gas is preferably high-purity nitrogen generated by a nitrogen generator, and the purity of the high-purity nitrogen is preferably 99.999%; the collision gas is preferably high-purity argon, and the purity of the high-purity argon is preferably 99.999%; before use, the flow rates of the atomization gas, the conical hole back blowing gas and the collision gas are preferably adjusted so that the sensitivity of the mass spectrum meets the detection requirement; voltage values such as taper hole voltage and collision energy are optimized to optimal sensitivity; the qualitative ion pair, the quantitative ion pair, the cone-hole voltage and the collision energy are shown in table 2.
TABLE 22, 3-dibromoacrylamide and 13 C 3 mass spectrometry parameters of (E) -2, 3-dibromoacrylamide
In the present invention, during the qualitative analysis, the mass spectrum qualitative ion of each test compound must appear, and should include at least one parent ion and two daughter ions, and the relative abundance ratio of the two daughter ions of 2, 3-dibromoacrylamide in the same acrylamide and the sample solution in the same test batch is compared with that of the standard solution with the same concentration, and the allowable deviation thereof is not more than the range specified in table 3:
TABLE 3 maximum permissible deviation of relative ion abundance in qualitative terms
| Relative ion abundance/%) | ＞50 | 20-50 | 10-20 | ≤10 |
| Allowable relative deviation/%) | ±20 | ±25 | ±30 | ±50 |
After a sample chromatogram is obtained, the invention obtains 2, 3-dibromoacrylamide and 13 C 3 -peak area of 2, 3-dibromoacrylamide; calculating the concentration of the 2, 3-dibromoacrylamide in the sample liquid to be detected by adopting an internal standard method according to the relative peak area and the linear curve, and calculating the content of the 2, 3-dibromoacrylamide in the sample liquid to be detected according to the formula (1); the linear curve is a linear curve of the chromatographic relative peak area of the 2, 3-dibromoacrylamide and the concentration of the acrylamide; the amount of the 2, 3-dibromoacrylamide is calculated by acrylamide.
In the present invention, the preparation of the linear curve preferably comprises the following steps:
preparing acrylamide series standard working solution;
and performing LC-MS/MS detection on the acrylamide series standard working solution to obtain a linear curve of the chromatographic relative peak area and the acrylamide concentration of the 2, 3-dibromoacrylamide.
The invention prepares acrylamide series standard working solution.
In the present invention, the method for preparing the acrylamide series standard working solution preferably comprises:
(1) preparing 1.0mg/mL acrylamide standard stock solution;
(2) preparing a standard acrylamide stock solution of 10 mg/L;
(3) preparing 1.0mg/L acrylamide standard use solution;
(4) prepared into a solution of 0.5mg/mL 13 C 3 -an acrylamide stock solution;
(5) to prepare 1.0mg/L 13 C 3 -an acrylamide stock solution;
(6) preparing acrylamide series standard working solution.
In the embodiment of the invention, the preparation of the 1.0mg/mL acrylamide standard stock solution specifically comprises: accurately weighing 10mg (accurate to 0.01mg) of acrylamide standard substance, dissolving in methanol in a 10mL brown volumetric flask, fixing the volume to the scale, and storing in dark and sealed condition at-20 ℃.
In the embodiment of the invention, the preparation of the standard acrylamide stock solution of 10mg/L specifically comprises the following steps: accurately sucking 0.25mL of 1.0mg/mL acrylamide standard stock solution, placing the stock solution into a 25mL brown volumetric flask, metering the volume of methanol to a scale, and storing the stock solution in a dark and sealed manner at the temperature of minus 20 ℃.
In the embodiment of the invention, the 1.0mg/L acrylamide standard use solution is prepared by the following specific steps: accurately sucking 1.00mL of 10mg/L acrylamide standard stock solution, placing the acrylamide standard stock solution into a 10mL brown volumetric flask, and fixing the volume of 0.1% formic acid aqueous solution to a scale, wherein the 10mg/L acrylamide standard stock solution is preferably prepared at the present.
In the examples of the present invention, the amount of 0.5mg/mL 13 C 3 -acrylamide stock solutionThe preparation method specifically comprises the following steps: accurately weighing 13 C 3 5.0mg (to the accuracy of 0.01mg) of acrylamide standard substance, dissolving in methanol and fixing to the scale in a 10mL brown volumetric flask, and storing in a dark and sealed condition at-20 ℃.
In the examples of the present invention, said 1.0mg/L 13 C 3 The preparation of the acrylamide stock solution is specifically: accurate suction of 0.1mg/mL 13 C 3 Placing the acrylamide stock solution into a 50mL brown volumetric flask, metering the volume of methanol to the scale, and storing the solution in a dark and sealed manner at 4 ℃.
In the embodiment of the invention, the preparation of the acrylamide series standard working solution specifically comprises the following steps: 10 μ L, 20 μ L, 50 μ L and 100 μ L of acrylamide standard use solution with a concentration of 0.1 μ g/mL, 20 μ L, 50 μ L and 100 μ L of acrylamide standard use solution with a concentration of 1.0 μ g/mL, 20 μ L, 50 μ L and 100 μ L of acrylamide standard use solution with a concentration of 10.0 μ g/mL are respectively added into 10 plastic centrifuge tubes, and then 20 μ L of acrylamide standard use solution with a concentration of 5.0 μ g/mL is respectively added into 10 plastic centrifuge tubes 13 C 3 And (3) deriving an acrylamide internal standard application solution and 5mL of water to obtain a 2, 3-dibromoacrylamide series standard working solution.
In the invention, the concentration of acrylamide in the 2, 3-dibromoacrylamide series standard working solution is 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, 50.0ng/mL, 100.0ng/mL, 200.0ng/mL, 500.0ng/mL and 1000.0ng/mL in sequence, 13 C 3 the concentration of acrylamide is 100 ng/mL. In the present invention, the kind, the amount and the derivatization condition of the reagent added during the derivatization process are preferably the same as those of the derivatization during the preparation process of the sample solution to be tested in the above technical solution, and are not described herein again.
After acrylamide series standard working solutions are subjected to LC-MS/MS detection, a linear curve of a chromatographic relative peak area and acrylamide concentration of 2, 3-dibromoacrylamide is obtained.
In the present invention, the detection conditions of LC-MS/MS are preferably the same as the conditions of liquid chromatography detection and mass spectrometry detection of the sample to be detected, and are not described herein again.
In the present invention, the LC-MS/MS detection is preferably performed from low concentration to high concentration in order of chromatographic peak area/internal standard of the target compound (2, 3-dibromoacrylamide) ((2, 3-dibromoacrylamide)) 13 C 3 -2, 3-dibromoacrylamide) with the chromatographic peak surface on the ordinate (Y) and the (acrylamide) concentration on the abscissa (X) to plot a linear curve: y is 2.54168X +0.272129 and r is 0.9997. In the invention, the linear range of the linear curve is preferably that the concentration of acrylamide is 1.0-1000.0 ng/mL, 13 C 3 acrylamide concentration of 100 ng/mL).
In the present invention, the response value of the sample solution to be measured is in the linear range of the linear curve, and when the response value exceeds the upper limit of the linear range, the response value is preferably determined again after the sample volume is reduced.
In the present invention, the formula (1) is as follows:
in the formula (1), X is the content of acrylamide in a sample and has a unit of mu g/kg;
c is the corresponding concentration of acrylamide in the sample in a linear curve according to an internal standard method, and the unit is ng/mL;
v is the constant volume of the liquid to be detected of the sample, and the unit is mL;
m is the sample mass in g;
k is the dilution factor.
When acrylamide is directly detected by liquid chromatography-tandem mass spectrometry, due to the strong polarity, the acrylamide is weakly retained on a general C18 chromatographic column, a chromatographic column with strong retention capacity (such as a T3 column) is required to be used for chromatographic separation, and due to the small molecular weight (MW 71), the sensitivity of ion fragments 72>44 is very low, so that the qualitative accuracy of low concentration of the acrylamide is influenced. The invention adopts bromine to generate 2, 3-dibromopropionamide, greatly reduces the polarity of the 2, 3-dibromopropionamide, can be well reserved even on a common C18 chromatographic column, and greatly improves the sensitivity of qualitative ions by adopting isotope difference parent ions.
According to the detection method provided by the invention, in the detection of the grain product sample, after water extraction and dichloromethane degreasing are adopted, the acrylamide content in the grain product sample can be accurately detected by direct derivation without further purification, and compared with the method of GB5009.204-2014 (determination of acrylamide in food safety national standard food), the detection method saves the cost, reduces the workload and is more environment-friendly. Particularly in the coffee sample detection process, the first method of GB5009.204-2014 determination of acrylamide in national food safety standards adopts a non-derivatization method to directly carry out liquid chromatography-tandem mass spectrometry to detect acrylamide in coffee, and due to the fact that the molecular weight is small and the retention capacity is weak, matrix interference is too large, and accurate determination cannot be carried out. After the sample is subjected to derivatization, acrylamide generates 2, 3-dibromoacrylamide, the molecular weight is greatly increased, the polarity is weakened, the retention capacity is enhanced, the influence of matrix interference is greatly reduced, and the target compound acrylamide can be effectively detected.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) Preparation of acrylamide series standard working solution
(1.1) preparation of 1.0mg/mL acrylamide Standard stock solution
Accurately weighing 10mg (accurate to 0.01mg) of acrylamide standard substance, dissolving in methanol in a 10mL brown volumetric flask, fixing the volume to the scale, and storing in dark and sealed condition at-20 ℃.
(1.2) preparing a standard stock solution of acrylamide at 10mg/L
Accurately sucking 0.25mL of 1.0mg/mL acrylamide standard stock solution, placing the solution in a 25mL brown volumetric flask, metering the volume of methanol to a scale, and storing the solution in a dark and sealed manner at the temperature of minus 20 ℃.
(1.3) preparation of 1.0mg/L acrylamide Standard solution for use
Accurately sucking 1.00mL of 10mg/L acrylamide standard stock solution, placing the acrylamide standard stock solution into a 10mL brown volumetric flask, and fixing the volume of 0.1% formic acid aqueous solution to a scale, wherein the 10mg/L acrylamide standard stock solution is preferably prepared at the present.
(1.4) preparation of 0.5mg/mL 13 C 3 -acrylamide stock solution
Accurately weighing 13 C 3 5.0mg (to the accuracy of 0.01mg) of acrylamide standard substance, dissolving in methanol and fixing to the scale in a 10mL brown volumetric flask, and storing in a dark and sealed condition at-20 ℃.
(1.5) preparation of 1.0mg/L 13 C 3 -acrylamide stock solution
Accurate suction of 0.1mg/mL 13 C 3 Placing the acrylamide stock solution into a 50mL brown volumetric flask, metering the volume of methanol to the scale, and storing the solution in a dark and sealed manner at 4 ℃.
(1.6) preparing acrylamide series standard working solution
10 mu.L, 20 mu.L, 50 mu.L and 100 mu.L of acrylamide quasi-use solution with the concentration of 0.1 mu g/mL, 20 mu.L, 50 mu.L and 100 mu.L of acrylamide standard use solution with the concentration of 1.0 mu g/mL, 20 mu.L, 50 mu.L and 100 mu.L of acrylamide standard use solution with the concentration of 10.0 mu g/mL are respectively added into 10 plastic centrifuge tubes, and then 20 mu.L of acrylamide standard use solution with the concentration of 5.0 mu g/mL is respectively added into 10 plastic centrifuge tubes 13 C 3 -acrylamide internal standard application liquid, 5mL of water and 0.75g of potassium bromide are dissolved in a vortex mode, then 0.3mL of sulfuric acid solution with the concentration of 0.3mol/L and 0.5mL of potassium bromate solution with the concentration of 17g/L are added, the mixture is evenly mixed after being covered, the mixture is kept in a dark place for derivatization for 30min, 0.05mL of sodium thiosulfate solution with the concentration of 0.25g/mL is added for removing redundant bromine, 3mL of ethyl acetate is added for vortex extraction for 5min, 10000r/min is centrifuged for 3min, obtained supernatant liquid is blown to be dry, 1.0mL of 0.1% formic acid aqueous solution is added for dissolution, 10000r/min is centrifuged for 3min, obtained supernatant liquid is 2, 3-dibromoacrylamide series standard working solutions, wherein the corresponding acrylamide concentrations are 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, 50.0ng/mL, 100.0ng/mL, 200.0ng/mL, 500.0ng/mL, 1000.0ng/mLng/mL， 13 C 3 The concentration of acrylamide is 100 ng/mL.
(2) Linear curve regression equation
Performing LC-MS/MS detection on the acrylamide series standard working solution from small to large according to the concentration to obtain the peak area of the 2, 3-dibromoacrylamide with corresponding concentration, 13 C 3 Peak area of 2, 3-dibromoacrylamide, area of the chromatographic peak of 2, 3-dibromoacrylamide- 13 C 3 And drawing a linear curve by taking the chromatographic peak surface of the 2, 3-dibromoacrylamide as an ordinate (Y) and taking the concentration of the acrylamide as an abscissa (X), so as to obtain a regression equation Y of the linear curve, wherein the regression equation Y is 2.54168X +0.272129, and r is 0.9997.
The conditions of the liquid chromatography detection in the liquid chromatography tandem mass spectrometry detection are as follows: the chromatographic column is a UPLC BEH C18 column (1.7 μm, 2.1 mm. times.100 mm); the column temperature is 30 ℃; the sample injection amount is 10 mu L; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.1% formic acid aqueous solution in percentage by volume, and the mobile phase B is a 0.1% formic acid methanol solution in percentage by volume; the flow rate of the mobile phase is 0.3 mL/min; a detector triple quadrupole mass spectrometry detector; the elution mode is gradient elution; the conditions of the gradient elution are shown in table 1.
TABLE 1 liquid chromatography gradient elution conditions
| Time/min | Mobile phase A/volume% | Mobile phase B/volume% |
| 0.00 | 95 | 5 |
| 3.00 | 95 | 5 |
| 5.00 | 85 | 15 |
| 5.10 | 0 | 100 |
| 6.00 | 0 | 100 |
| 6.10 | 95 | 5 |
| 7.50 | 95 | 5 |
The conditions of mass spectrum detection in the liquid chromatography tandem mass spectrum detection comprise: the ion source is an electrospray ion source (ESI +); the ion source temperature is 150 ℃; the capillary voltage is 3.0 kV; the desolventizing temperature is 500 ℃; the scanning mode is positive ion scanning; the detection mode is multi-reaction detection (MRM); the atomizing gas is high-purity nitrogen with the purity of 99.999 percent; the back blowing of the taper hole is high-purity nitrogen with the purity of 99.999 percent; the collision gas is high-purity argon with the purity of 99.999 percent; before use, the flow rates of the atomized gas, the cone hole back blowing gas and the collision gas are adjusted to enable the sensitivity of the mass spectrum to meet the detection requirement; voltage values such as taper hole voltage and collision energy are optimized to the optimal sensitivity; the qualitative ion pair, the quantitative ion pair, the cone-hole voltage and the collision energy are shown in table 2, and the standard map is shown in fig. 1.
TABLE 22, 3-dibromoacryloylAmines and 13 C 3 mass spectrometry parameters of (E) -2, 3-dibromoacrylamide
Example 2
(1) Sample pretreatment
(1.1) crushing the potato chips by a crusher, accurately weighing 0.5g (to be accurate to 0.0001g) of crushed potato chip samples in a 25mL graduated centrifuge tube, and adding 40 mu L of potato chips with the concentration of 5.0 mu g/L 13 C 3 Ultrasonically extracting an acrylamide internal standard application liquid and 10mL of ultrapure water for 10min, adding 5mL of dichloromethane for degreasing, performing vortex for 5min, centrifuging at 10000r/min for 3min, and taking out a supernatant, namely an extracting solution;
(1.2) purifying 5.0mL of the sample extracting solution in a GCB/PSA column, wherein the flow rate of the sample extracting solution is 2mL/min, and collecting the sample purifying solution to a 15mL graduated centrifuge tube;
(1.3) adding 0.75g of potassium bromide into 5mL of sample purifying solution, performing vortex dissolution, adding 0.3mL of sulfuric acid solution with the concentration of 0.3mol/L and 0.5mL of potassium bromate solution with the mass volume percentage concentration of 1.7%, capping, uniformly mixing, standing in a dark place for derivatization for 30min, adding 0.05mL of sodium thiosulfate solution with the mass volume percentage concentration of 25% to remove redundant bromine, adding 3mL of ethyl acetate, performing vortex extraction for 5min, performing centrifugation for 3min at 10000r/min, taking supernatant nitrogen for blow-drying, adding 1.0mL of formic acid solution with the concentration of 0.1% (volume percentage content) for dissolution, and performing centrifugation for 3min at 10000r/min to obtain supernatant, namely the sample solution to be detected;
(2) quantitative analysis
Performing LC-MS/MS detection on the sample liquid to be detected by adopting the conditions of liquid phase detection chromatography and mass spectrum detection in the embodiment 1 to obtain a sample chromatogram, and obtaining the relative peak area of 2, 3-dibromoacrylamide in the sample according to the sample chromatogram; and (3) calculating the content of the 2, 3-dibromoacrylamide (calculated by acrylamide) in the sample liquid to be detected according to the formula (1) and the regression equation of the standard curve obtained in the example 1.
(3) Qualitative analysis
The mass spectrometric qualitative ion of each test compound must be present, at least one parent ion and two daughter ions, and the relative abundance ratio of the two daughter ions of 2, 3-dibromoacrylamide in the same sample in the same test batch, for the same acrylamide, compared to a standard solution of comparable concentration, must not deviate by more than the range specified in table 3:
TABLE 3 maximum permissible deviation of relative ion abundance in qualitative terms
| Relative ion abundance/%) | ＞50 | 20-50 | 10-20 | ≤10 |
| Allowable relative deviation/%) | ±20 | ±25 | ±30 | ±50 |
The ion ratio was within. + -. 20%, and acrylamide was judged to be positive.
The TIC spectrum of the potato chip sample is shown in fig. 2, and as can be seen from fig. 2, the acrylamide content in the potato chip is 294.3 μ g/kg (n is 3).
Example 3
The detection was carried out according to the method of example 2, which differs from example 2 in that: the standard concentration of acrylamide in the sample solution to be tested is 100 mug/kg, the test result is the average value of 6 experiments, and the test result is shown in table 4.
Example 4
The detection was carried out according to the method of example 2, which differs from example 2 in that: the standard concentration of acrylamide in the sample solution to be tested is 500 mug/kg, the test result is the average value of 6 experiments, and the test result is shown in table 4.
Example 5
The detection was carried out according to the method of example 2, which differs from example 2 in that: the standard concentration of acrylamide in the sample solution to be tested is 1000 mug/kg, the test result is the average value of 6 experiments, and the test result is shown in table 4.
Example 6
The detection was carried out according to the method of example 2, differing from example 2 in that: the sample is coffee, the standard concentration of acrylamide in the sample liquid to be tested is 300 mug/kg, the test result is the average value of 6 experiments, and the test result is shown in table 4.
The TIC spectrum of the coffee sample is shown in FIG. 3. As can be seen from FIG. 3, the coffee sample contains acrylamide.
Example 7
The detection was carried out according to the method of example 2, differing from example 2 in that: the sample is coffee, the standard concentration of acrylamide in the sample liquid to be tested is 800 mug/kg, the test result is the average value of 6 experiments, and the test result is shown in table 4.
TABLE 4 recovery results for acrylamide in potato chips and coffee extracts using standard addition
As shown in Table 4, the acrylamide concentrations in the potato chips and coffee samples were 100-1000. mu.g/kg, the average recovery rates were 87.4-111.2%, and the accuracies were 2.6-7.6%. When 0.5g of sample is taken, the limit of quantification of acrylamide is 10.0 mug/kg (the limit of quantification is calculated by 10 times of signal-to-noise ratio), and the limit of detection is 3.0 mug/kg (the limit of detection is calculated by 3 times of signal-to-noise ratio), which shows that the detection method provided by the invention has high accuracy and precision.
Example 8
The purchased potato chips and coffee samples are detected according to the method of the embodiment 2, wherein the pretreatment method of the coffee comprises the following steps (1.1): pulverizing coffee with pulverizer, accurately weighing pulverized coffee sample 0.2g (accurate to 0.0001g) in 50mL graduated centrifuge tube, adding 80 μ L of 5.0 μ g/L 13 C 3 And (3) carrying out vortex dissolution on the acrylamide internal standard application liquid and 20mL of ultrapure water, adding 5mL of dichloromethane for degreasing, carrying out vortex for 5min, centrifuging at 10000r/min for 3min, and taking out the supernatant, namely the extracting solution. The results are shown in table 5:
TABLE 5 detection results of acrylamide in different potato chip and coffee samples
As can be seen from Table 5, the collected potato chips and coffee both contain a certain amount of acrylamide and have higher contents of a part of acrylamide, and the pretreatment method and the detection method provided by the invention can completely meet the detection requirements of actual samples.
Example 9
The FAPAS quality control sample biscuits are measured according to the method of example 2 (the code is T3094QC, the quality control sample is assigned 293 mu g/kg, and the content range is 181-406 mu g/kg), and the results are 298.2 mu g/kg, 293.6 mu g/kg and 291.1 mu g/kg after independent measurement for 3 times, the average result is 294.3 mu g/kg, the recovery rate is 100.4%, and the accuracy is high.
Comparative example 1
Pretreating and detecting a coffee sample according to a method of GB5009.204-2004, wherein the peak-off time of acrylamide is 1.7 min; under the condition that the flow rate of 0.3mL/min of 0.1% formic acid water-methanol (volume ratio is 90:10), the peak-out time of acrylamide is 1.7min, the chromatogram is shown in FIG. 4, and the top to bottom in FIG. 4 are as follows: acrylamide 72>55 ion channels in the sample, acrylamide 72>44 ion channels in the sample, total acrylamide ion spectra in the sample, acrylamide 72>55 ion channels in the standard, acrylamide 72>44 ion channels in the standard and total acrylamide ion spectra in the standard.
As can be seen from FIGS. 1 and 4, due to the small molecular weight of acrylamide (MW 71) and weak retention capacity (1.7min), the sensitivity of ion fragments 72>44 is very low, which affects the qualitative accuracy of its low concentration. According to the invention, acrylamide is derived to generate 2, 3-dibromopropionamide, so that the polarity of the acrylamide is greatly reduced, the acrylamide can be well retained even on a common C18 chromatographic column, and the sensitivity of qualitative ions is greatly improved by adopting isotope difference parent ions.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for detecting acrylamide in thermally processed food is characterized by comprising the following steps:
mixing the hot processed food sample, 13 C 3 -mixing an acrylamide internal standard application liquid with water for extraction, mixing the obtained extraction system with dichloromethane for degreasing to obtain a sample extraction liquid;
purifying the sample extracting solution by adopting a GCB/PSA column to obtain a sample purifying solution;
mixing the sample purifying solution, potassium bromide, a sulfuric acid solution and a potassium bromate solution, and deriving to obtain a sample solution to be detected;
detecting the content of the 2, 3-dibromopropionamide in the sample liquid to be detected by adopting a liquid chromatography tandem mass spectrum;
the amount of the 2, 3-dibromopropionamide is calculated by acrylamide;
the conditions of the liquid chromatography detection in the liquid chromatography tandem mass spectrometry detection comprise:
a chromatographic column: a UPLCBEHC18 column;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
mobile phase: the mobile phase A is a formic acid aqueous solution, and the volume percentage concentration of the formic acid aqueous solution is 0.05-0.3%; the mobile phase B is a methanoic acid solution, and the volume percentage concentration of the methanoic acid solution is 0.05-0.3%; the flow rate of the mobile phase is 0.2-0.6 mL/min;
and (3) an elution mode: gradient elution;
the procedure for the gradient elution was as follows:
0.00-3.00 min, wherein the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%;
3.00-5.00 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 85% at a constant speed, and the volume percentage of the mobile phase B is increased from 5% to 15% at a constant speed;
5.00-5.10 min, wherein the volume percentage of the mobile phase A is reduced from 85% to 0% at a constant speed, and the volume percentage of the mobile phase B is increased from 15% to 100% at a constant speed;
5.10-6.00 min, wherein the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is 100%;
the volume percentage of the mobile phase A is increased from 0% to 95% at a constant speed and the volume percentage of the mobile phase B is decreased from 100% to 5% at a constant speed for 6.00-6.10 min;
and 6.10-7.50 min, wherein the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%.
2. The detection method according to claim 1, wherein the extraction manner comprises ultrasonic extraction or vortex extraction.
3. The assay of claim 1, wherein the thermally processed food sample comprises a food product, coffee beans, or a coffee beverage.
4. The detection method according to claim 1 or 3, wherein the ratio between the mass of the sample of thermally processed food and the volume of water is 1 g: (5-100) mL.
5. Detection method according to claim 1 or 3, characterised in that the ratio between the mass of the thermally processed food sample and the volume of dichloromethane is 1 g: (5-25) mL.
6. The detection method according to claim 1, wherein the molar ratio of the potassium bromate solution to the potassium bromide solution to the sulfuric acid solution is 1: (5-6): (3-4).
7. The detection method according to claim 1, wherein the derivatization is performed under sealed and light-shielded conditions.
8. The detection method according to claim 1, wherein the conditions for mass spectrometry detection in the liquid chromatography tandem mass spectrometry detection comprise:
an ion source: an electrospray ion source;
ion source temperature: 150 ℃;
capillary voltage: 3.0 kV;
desolventizing temperature: 500 ℃;
the scanning mode is as follows: scanning positive ions;
the detection mode is as follows: detecting multiple reactions;
atomizing: nitrogen gas or high purity nitrogen gas produced by a nitrogen generator;
back blowing of the taper hole: nitrogen gas or high purity nitrogen gas produced by a nitrogen generator;
collision gas: high purity argon gas.
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