CN113155989A - Rapid detection method for acrylamide content in tea - Google Patents
Rapid detection method for acrylamide content in tea Download PDFInfo
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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
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- 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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- 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
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- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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Abstract
The invention discloses a rapid detection method of acrylamide content in tea, which is operated according to the following steps: s1, establishing an internal standard curve: weighing samples, and measuring the acrylamide content of each concentration by taking one concentration in a series of acrylamide standard solutions as an addition sample; is prepared from acrylamide and13C3]-the concentration ratio of the standard solution of acrylamide isotope is abscissa, and linear regression analysis is performed by taking the peak area ratios corresponding to mass-to-charge ratios m/z of 72 and 75 as ordinate to obtain a standard curve; s2, sample pretreatment: weighing a sample, and sequentially adding isotope-labeled acrylamide standard solutionPurified water, acetonitrile, anhydrous magnesium sulfate and sodium chloride, and vortex oscillation is carried out when the reagent is added; placing in a water bath environment for ultrasonic and centrifugal treatment, taking out acetonitrile, adding PSA (pressure swing adsorption) for full oscillation and centrifugation, taking supernatant, blowing nitrogen for concentration and drying, adding distilled water for redissolution, and filtering to obtain a pretreatment sample; s3, sample detection: and (4) detecting the pre-treated sample, and calculating the content of acrylamide by an internal standard method.
Description
Technical Field
The invention relates to the technical field of agricultural product detection, in particular to a rapid detection method for the acrylamide content in tea.
Background
Animal experiments have shown that acrylamide (AA) has liver and neurotoxic, reproductive toxicity, and is carcinogenic to rodents, listed as a "possible human carcinogen" by the international agency for research on cancer (IARC) in 1994. In 4 months in 2002, Swedish scientists find that a large amount of acrylamide can be generated in the thermal processing process of starch food, which causes wide social concern, and academia carries out a series of researches on the acrylamide in the food. In all these studies, the acrylamide content of food products cannot be measured. However, since there are many kinds of foods, the matrix composition is very complicated, and the content of acrylamide varies greatly depending on the processing method of different kinds of foods, a single sample pretreatment method cannot be applied to all foods. Therefore, it is necessary to establish corresponding detection methods for different types of food. QuEChERS (quick, easy, chemical, effective, rugged and safe) technology was developed by the American Ministry of agriculture in the detection of pesticide residues.
China is a world-recognized tea origin and has a long tea culture history. Meanwhile, China is also a large tea-producing country, and tea is an important economic crop in China. Tea is taken as one of three beverages in the world, and is popular among people because of having the effects of clearing heat, quenching thirst and various health-care effects. Researches show that in the processing processes of enzyme deactivation, fermentation and the like, the organic matrix components in the tea can generate complex chemical reactions, so that acrylamide can be generated. The matrix component of tea is mainly cellulose, and then contains organic components such as pigment, tea polyphenol, organic acid, caffeine, etc. For the detection of acrylamide in tea, a solid-phase extraction column (SPE column) is generally adopted for purification at present, but the whole process of SPE column treatment (activation, sample loading, elution and elution) needs 2-4 h and needs a specific device, the operation process is complex and time-consuming, and the rapid detection of the content of acrylamide in tea is not facilitated.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing detection method for the acrylamide content in the tea leaves is inconvenient to operate and consumes a long time.
In order to solve the technical problems, the invention provides a rapid detection method of the acrylamide content in tea, which comprises the following steps:
s1, establishing an internal standard curve: quantitatively weighing the crushed tea sample, and measuring the acrylamide and the alpha [ alpha ], [ beta ] and [ beta ] in the same kind of a [ beta ] or a [ beta ] in a series of acrylamide ] or [ beta ] in a series of acrylamide standard solution of acrylamide ] in a standard solution of acrylamide ] in the standard solution, and [ beta ] respectively13C3]-acrylamide content; then using a series of acrylamide standard substance solutions and13C3]-the acrylamide isotope standard solution concentration ratio is an abscissa, and linear regression analysis is performed to obtain a standard curve by taking peak area ratios corresponding to mass-to-charge ratios m/z of 72 and 75 as an ordinate respectively in an SIR selected ion scanning mode;
s2, sample pretreatment: quantitatively weighing a crushed tea sample, then sequentially adding an acrylamide standard solution marked by an isotope, purified water, acetonitrile, anhydrous magnesium sulfate and sodium chloride, and carrying out vortex oscillation for a certain time by adding one reagent each time; then placing the uniformly mixed reagent and sample in a water bath environment for ultrasonic treatment, then carrying out centrifugal treatment, taking out a proper amount of acetonitrile, adding PSA (N-propylethylenediamine) for full oscillation, then centrifuging again, then taking out supernatant, carrying out nitrogen blowing concentration to be nearly dry, adding distilled water for redissolving and filtering to obtain a pre-treated sample; acrylamide is a polar small-molecule organic compound that is readily soluble in water and polar organic solvents, water and acetonitrile being common extraction solvents. Anhydrous MgSO (MgSO)4And NaCl is added to increase the polarity of the aqueous solution and reduce organic impuritiesWhile the separation of acetonitrile and water is facilitated. The detection limit of acrylamide in a sample can be greatly improved by using acetonitrile as a solvent, because the acetonitrile is blown to be nearly dry by nitrogen after being extracted and is redissolved by a very small amount of water, so that the concentration of acrylamide in the sample amount is increased. Therefore, the method still adopts acetonitrile as an extraction solvent.
S3, sample detection: and (5) detecting the to-be-detected sample of the pretreatment sample prepared in the step (S2) by adopting a high performance liquid chromatography tandem mass spectrometry method, and calculating the content of acrylamide by an internal standard method.
Preferably, the regression equation of the standard curve in step S1 is y ═ 1.0624 x-0.0605, and R is20.9999, wherein x is acrylamide standard solution and [ 2 ]13C3]Concentration ratio of acrylamide isotope standard solution, y is peak area ratio corresponding to mass-to-charge ratio m/z of 72 and 75 respectively in selected ion scanning mode of SIR, R2The regression equation has a good linear relationship between acrylamide contents of 0.01 μ g/ml to 2.00 μ g/ml for standard deviation.
Preferably, the series of acrylamide standard solutions is prepared in the following manner:
s11, preparing an acrylamide stock solution: accurately weighing 0.1g of acrylamide standard substance, and fixing the volume to 100ml by using distilled water to prepare 1mg/ml acrylamide stock solution;
s12, preparing an acrylamide standard stock solution: taking 1ml of 1mg/ml acrylamide stock solution, and adding distilled water to a constant volume of 100ml to prepare 0.01mg/ml acrylamide standard stock solution;
s13, PREPARATION (MIFARE)13C3]-an internal standard solution of acrylamide isotope: taking 500. mu.l of 1mg/ml [ sic ], [ solution ]13C3]An acrylamide isotope solution prepared by diluting to 100ml with distilled water to 5. mu.g/ml13C3]-an acrylamide isotope stock solution;
s14, preparing an acrylamide standard solution: respectively taking 10, 50, 100, 500, 1000 and 2000 ul of acrylamide standard stock solution, and respectively adding 200 ul of the solution13C3]-acrylamide isotope internal standard solution, distilled water to constant volume of 10ml, and preparation concentration0.01, 0.05, 0.10, 0.50 and 2.00 mu g/ml of series acrylamide standard solutions.
Preferably, the isotope-labeled acrylamide standard solution in the step S2 is [1, 2, 3,13C3]standard solutions of acrylamide, i.e. where the carbon atoms in the 1, 2, 3 positions of the acrylamide carbon chain are isotopically substituted13C3And (4) marking.
Preferably, the ratio of the tea sample to the isotope-labeled acrylamide standard solution, purified water, acetonitrile, anhydrous magnesium sulfate and sodium chloride in the step S2 is 1 g: 150-240 μ l: 4-8 ml: 8-12 ml: 3-5 g: 0.5g, the concentration of the isotope-labeled acrylamide standard solution is 5 mug/ml. Wherein, the optimal dosage ratio of the tea sample to the isotope labeled acrylamide standard solution, purified water, acetonitrile, anhydrous magnesium sulfate and sodium chloride is 1 g: 200 microliter: 5 ml: 10 ml: 4 g: 0.5g of a mineral oil in the form of a mineral oil,
preferably, the vortex oscillation time in the step S2 is 2-5 minutes, the temperature of the water bath of the uniformly mixed reagent and sample is 36-42 ℃, the ultrasonic treatment time is 1-5 minutes, the rotation speed of the first centrifugal treatment is 3500-4200 r/min, and the centrifugal time is 6-10 minutes; the extraction amount of acetonitrile was 6ml, and the addition amount of PSA was 0.2 g; the rotating speed of the second centrifugal treatment is 3600-4500 r/min, and the centrifugal time is 6-12 minutes; the extraction amount of the supernatant is 5 ml; the temperature of nitrogen blowing in the step S2 is 45-55 ℃, and preferably 50 ℃.
Preferably, the optimal conditions of the first centrifugation in the step S2 are a rotation speed of 4000r/min and a centrifugation time of 8 minutes; the optimal conditions of the second centrifugal treatment are that the rotating speed is 4000r/min and the centrifugal time is 8 minutes.
Preferably, the amount of distilled water added during reconstitution in step S2 is 1ml, and the filtration membrane during filtration is a 0.22 μm aqueous filtration membrane.
Preferably, the conditions of the high performance liquid chromatography in step S3 are as follows: venusil MP C18 chromatography column (4.6mm x 250mm, 5 μm,
)；sample introduction amount: 20 mu l of the mixture; the flow rate is 0.6 mL/min; mobile phase: methanol-water (5: 95 by volume); time: for 10 min.
Preferably, the mass spectrometry conditions in step S3 are: the ionization mode is ESI+A mode; the ion source temperature is 100 ℃; the desolventizing temperature is 300 ℃; gas (N)2) Flow rate: the desolventizing gas is 350L/h, and the taper hole gas is 50L/h; capillary voltage: 3.0 KV; taper hole voltage: 20V, and (3); SIR selection ion mode; the acrylamide quantitative ion m/z is 72 [, ]13C3]The acrylamide isotopically quantified ion m/z is 75.
Compared with the prior art, the invention has the following advantages:
the rapid detection method of the acrylamide content in the tea leaves firstly passes13C3]An acrylamide isotope is used for making an internal standard curve for quantification, so that the accuracy of a measurement result and a higher recovery rate are ensured, and then the QuEChERS method is combined with a high performance liquid chromatography tandem mass spectrometry (HPLC-MS) method, so that the raw material of the tea powder is quickly pretreated, namely, a one-pot sample treatment mode is adopted, the pretreatment operation is simplified, the use amount of a solvent is reduced, and the method is economical and efficient; then separating out acrylamide by high performance liquid chromatography, and finally detecting the content of the acrylamide in the sample by ESI-MS (electrospray ionization mass spectrometry) with high detection sensitivity.
Drawings
FIG. 1 is a graph comparing the recovery rates of acrylamide measured by pretreatment with n-hexane and without n-hexane in the examples of the present invention, in which the shaded bar graph is the yield of acrylamide of a tea sample treated with n-hexane, the unshaded bar graph is the yield of acrylamide of a tea sample not treated with n-hexane, and the ordinate is the yield of acrylamide of a tea sample.
FIG. 2 is a graph showing the effect of ultrasonic extraction time on the efficiency of acrylamide extraction, wherein the abscissa represents the ultrasonic extraction time and the ordinate represents the acrylamide content.
FIG. 3 is a graph showing the effect of ultrasonic extraction temperature on the efficiency of acrylamide extraction, where the abscissa is the temperature of the water bath during the ultrasonic extraction.
FIG. 4 is a graph of acrylamide standard curve in which the abscissa represents the sum of the acrylamide standard solution and [ 2 ]13C3]-acrylamide isotope standard solution concentration ratio, ordinate is peak area ratio corresponding to mass-to-charge ratio m/z of 72 and 75, respectively, in SIR selected ion scanning mode.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example (b): as shown in fig. 1 to 4, this embodiment provides a method for rapidly detecting the acrylamide content in tea.
Materials, reagents and apparatus therefor
Materials and reagents: acrylamide (AA) standard (purity is more than or equal to 99.9%), Sigma company of America; [1,2,3,13C3]-acrylamide isotope internal standard, cambridge isotope laboratory; methanol: chromatographic pure, seoul, korea; water for experiment: drochen pure water; acetonitrile: analytical purity, chemical reagents of national drug group limited; n-propylethylenediamine: analytically pure, Shanghai' an spectral experiment science and technology company; anhydrous magnesium sulfate: analytical purity, Kemi European Chemicals Limited company; sodium chloride: analytically pure, Guangzhou chemical reagent plant.
Commercial tea samples: green tea from Anhui and Henan, oolong tea from Guangdong and Fujian, black tea from Fujian and Anhui, and Pu' er tea from Yunnan.
Equipment: 1525 high performance liquid chromatograph, ZQ2000 single quadrupole mass spectrometer: waters corporation, USA; low-speed automatic balancing centrifuge: Shanghai-Hengchang scientific instruments, Inc.; UTN-2800D Nitrogen lance: tianjin Autt Sprens instruments, Inc.
Carrying out the process
This example sets up a calibration curve for a sample of tea leaves from Fujian black tea by first crushing black tea powder into powder, then dividing the black tea powder into 5 portions by taking 5g aliquots, adding 200. mu.l of a 0.01. mu.g/ml acrylamide standard solution to the first sample, 200. mu.l of a 0.05. mu.g/ml acrylamide standard solution to the second sample, 200. mu.l of a 0.10. mu.g/ml acrylamide standard solution to the third sample, 200. mu.l of a 0.50. mu.g/ml acrylamide standard solution to the fourth sample, and 200. mu.l of a 2.0. mu.g/ml acrylamide standard solution to the fifth sample.
Then, 5ml of purified water, 10ml of acetonitrile, 4g of anhydrous magnesium sulfate and 0.5g of sodium chloride are sequentially added into five tea leaf samples respectively, and each reagent is added for vortex oscillation for 2 min; then placing the uniformly mixed reagent and sample in a water bath environment at 39-41 ℃ for ultrasonic treatment for 2min, and then performing centrifugal treatment, wherein the rotating speed of a centrifugal machine is 4000r/min, and the centrifugal time is 8 min; then 6ml of acetonitrile is taken out, 0.2g of PSA is added, the mixture is fully oscillated and centrifuged again, the rotating speed of a centrifuge is 4000r/min, and the centrifuging time is 8 minutes; then 5ml of the supernatant was taken out and concentrated to near dryness by nitrogen blowing at 50 ℃, and then 1ml of distilled water was added to redissolve and filtered with a 0.22 μm aqueous filter membrane to obtain a pretreated sample. And then, inputting the five pre-processed samples into a high performance liquid chromatograph and a mass spectrometer respectively for separation and mass spectrometry, wherein the high performance liquid chromatograph and the mass spectrometer are required to be cleaned once between each sample, and the influence of the last residual sample on the detection result of the next sample is avoided. The conditions of the high performance liquid chromatography are as follows: venusil MP C18 chromatography column (4.6mm x 250mm, 5 μm,
) (ii) a Sample introduction amount: 20 mu l of the mixture; the flow rate is 0.6 mL/min; mobile phase: methanol-water (5: 95 by volume); time: for 10 min. The mass spectrum conditions are as follows: the ionization mode is ESI+A mode; the ion source temperature is 100 ℃; the desolventizing temperature is 300 ℃; gas (N)2) The flow rate is 350L/h of desolventizing gas and 50L/h of taper hole gas; capillary voltage: 3.0 KV; taper hole voltage: 20V, and (3); SIR selection ion mode; the acrylamide quantitative ion m/z is 72 [, ]13C3]-acrylamide isotopeThe quantitative ion m/z was 75.
Each set of experiments was repeated 3 times, plotted using Microsoft Excel software, and analyzed for variance using SPSS 19.0 software with a significance level of 0.05, yielding the standard curve of fig. 4. The measurement of 8 tea samples to be measured is carried out according to the method so as to calculate the content of acrylamide in the samples according to the mass spectrum area of the measured acrylamide, and the content of acrylamide in the 8 tea samples is obtained as shown in table 1:
table 1: 8 tea samples and acrylamide content thereof
According to the characteristics of the matrix components of the tea, the invention establishes a method for rapidly detecting the content of acrylamide in the tea by QuEChERS pretreatment and HPLC-MS separation. QuEChERS realizes the sample pretreatment mode of 'one pot boiling', the HPLC separation is rapid and efficient, the ESI-MS (electrospray ionization mass spectrometry) detection sensitivity is high, and simultaneously the method uses13C3]And an acrylamide isotope is used for making an internal standard curve for quantification, so that the accuracy of the measurement result and higher recovery rate are ensured. The method simplifies the pretreatment operation, reduces the use of organic solvents, has the characteristics of simplicity, rapidness, high sensitivity and good reproducibility, and is suitable for measuring the content of acrylamide in tea leaves.
Influencing factor
1. Selection of sample size
Experiments were carried out on Fujian black tea samples with weights of 0.5g, 1g and 1.5g, and the effect of sample amount on recovery rate was examined by using a sample standard recovery rate method. The loading level for each sample was 20. mu.g/kg, versus acrylamide recovery (as shown in Table 2). As a result, it was found that the recovery rate was the highest for the sample having a weight of 1 g. Due to different processing technologies of various tea leaves, the content of acrylamide is different, too small sample amount can cause that the content of the acrylamide is possibly lower than the quantitative detection Limit (LOQ) of the method and cannot be quantified, and the matrix effect caused by too large sample amount can influence the accuracy of the determination result. Thus, a sample weight of 1g has broader applicability and representativeness.
Table 2: acrylamide content and recovery rate of samples with different weights
2. Effect of n-Hexane
In order to eliminate the influence of lipids in food matrix components on the measurement, many studies on the detection of acrylamide in foods have been carried out by degreasing with n-hexane. Although the content of lipid components in the tea is low, the tea contains a plurality of organic components such as pigments, and in theory, normal hexane can also be used for removing organic interference impurities. The invention compares the influence of the normal hexane used in the pretreatment and the normal hexane not used as the degreasing agent on the recovery rate of the acrylamide (as shown in figure 1), and the result shows that the recovery rate of the acrylamide is reduced to different degrees after the normal hexane treatment. In addition, the use of n-hexane in the pretreatment increases the use of organic solvents, and the pretreatment operation of the sample is more complicated. Therefore, n-hexane is not used as the degreasing agent in the invention.
3. Influence of ultrasonic extraction
The ultrasonic wave has mechanical effect, cavitation effect and thermal effect, can increase the moving speed of medium molecules, increase the penetrating power of the medium and is beneficial to the dissolution of substances. The ultrasonic extraction operation is simple, and the ultrasonic extraction method is often used for extracting and separating active ingredients of natural products. The temperature of the water bath and the extraction time in the ultrasonic extraction are taken as single factors, and the influence of the temperature and the extraction time on the extraction rate of the acrylamide is examined. As can be seen from the attached figure 2, the extraction rate of the acrylamide is obviously improved after the ultrasonic extraction, the extraction time reaches the maximum after 2 minutes, and the extraction rate of the acrylamide is not obviously changed after the extraction time is continuously prolonged. The ultrasonic extraction was carried out for 2 minutes by gradually increasing the temperature of the water bath from room temperature, and as a result, it was found that the extraction rate of acrylamide reached a maximum at 40 ℃ and then slightly decreased as the temperature of the water bath was increased (as shown in FIG. 3). Therefore, the optimal extraction conditions were selected to be ultrasonic in a water bath at 40 ℃ for 2 minutes.
In summary, the rapid detection method of the acrylamide content in the tea leaves firstly passes the [ 2 ]13C3]An acrylamide isotope is used for making an internal standard curve for quantification, so that the accuracy of a measurement result and a higher recovery rate are ensured, and then the QuEChERS method is combined with a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method, so that the tea powder raw material is quickly pretreated, namely, a one-pot sample treatment mode is adopted, the pretreatment operation is simplified, the use amount of a solvent is reduced, and the method is economical and efficient; then separating out acrylamide through high performance liquid chromatography, and finally detecting the content of the acrylamide in the sample through ESI-MS (electrospray ionization mass spectrometry) with high detection sensitivity.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of the present invention, and these modifications and substitutions should also be regarded as the protection scope of the present invention.
Claims (10)
1. A rapid detection method for the acrylamide content in tea is characterized by comprising the following steps:
s1, establishing an internal standard curve: quantitatively weighing the crushed tea sample, and measuring the acrylamide and the alpha [ alpha ], [ beta ] and [ beta ] in the same kind of a [ beta ] or a [ beta ] in a series of acrylamide ] or [ beta ] in a series of acrylamide standard solution of acrylamide ] in a standard solution of acrylamide ] in the standard solution, and [ beta ] respectively13C3]-acrylamide content; then using a series of acrylamide standard substance solutions and13C3]-the concentration ratio of the acrylamide isotope standard solution is an abscissa, and linear regression analysis is performed to obtain a standard curve by taking peak area ratios corresponding to mass-to-charge ratios m/z of 72 and 75 as an ordinate respectively in an SIR selected ion scanning mode;
s2, sample pretreatment: quantitatively weighing a crushed tea sample, then sequentially adding an isotope-labeled acrylamide standard solution, purified water, acetonitrile, anhydrous magnesium sulfate and sodium chloride, and performing vortex oscillation for a certain time by adding one reagent each time; then placing the uniformly mixed reagent and sample in a water bath environment for ultrasonic treatment, then carrying out centrifugal treatment, taking out a proper amount of acetonitrile, adding PSA (pressure swing adsorption) for full oscillation, then centrifuging again, then taking out supernatant, carrying out nitrogen blowing concentration until the supernatant is nearly dry, adding distilled water for redissolving and filtering to obtain a pre-treated sample;
s3, sample detection: and (5) detecting the to-be-detected sample of the pretreatment sample prepared in the step (S2) by adopting a high performance liquid chromatography tandem mass spectrometry method, and calculating the content of acrylamide by an internal standard method.
2. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: the regression equation of the standard curve in the step S1 is that y is 1.0624 x-0.0605, and R is20.9999, wherein x is acrylamide standard solution and [ 2 ]13C3]-acrylamide isotope standard solution concentration ratio, y is peak area ratio corresponding to mass-to-charge ratio m/z of 72 and 75 respectively in SIR selected ion scanning mode, R2Is the standard deviation.
3. The method for rapidly detecting the acrylamide content in tea leaves as claimed in claim 1, wherein the series of acrylamide standard solutions in the step S1 is prepared as follows:
s11, preparing an acrylamide stock solution: accurately weighing 0.1g of acrylamide standard substance, and fixing the volume to 100ml by using distilled water to prepare 1mg/ml acrylamide stock solution;
s12, preparing an acrylamide standard stock solution: taking 1ml of 1mg/ml acrylamide stock solution, and adding distilled water to a constant volume of 100ml to prepare 0.01mg/ml acrylamide standard stock solution;
s13, PREPARATION (MIFARE)13C3]-an internal standard solution of acrylamide isotope: taking 500. mu.l of 1mg/ml [ sic ], [ solution ]13C3]An acrylamide isotope solution prepared by diluting to 100ml with distilled water to 5. mu.g/ml13C3]-an acrylamide isotope stock solution;
s14, preparing an acrylamide standard solution: taking 10, 50, 100, 500, 1000 and 2000 mul acrylamide standard stock solutions respectively200. mu.l of each of the two kinds of the additive was added13C3]Acrylamide isotope internal standard solution, distilled water to 10ml, and preparing serial acrylamide standard solutions with the concentrations of 0.01, 0.05, 0.10, 0.50 and 2.00 mu g/ml respectively.
4. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: the isotope-labeled acrylamide standard solution in the step S2 is a solution of [1, 2, 3,13C3]standard solutions of acrylamide, i.e. where the carbon atoms in the 1, 2 and 3 positions of the acrylamide carbon chain are isotopically substituted13C3And (4) marking.
5. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: in the step S2, the dosage ratio of the tea sample to the isotope-labeled acrylamide standard solution, purified water, acetonitrile, anhydrous magnesium sulfate and sodium chloride is 1 g: 150-240 μ l: 4-8 ml: 8-12 ml: 3-5 g: 0.5g, the concentration of the isotope-labeled acrylamide standard solution is 5 mug/ml.
6. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: the vortex oscillation time in the step S2 is 2-5 minutes, the temperature of the uniformly mixed reagent and sample water bath is 36-42 ℃, the ultrasonic treatment time is 1-5 minutes, the rotation speed of the first centrifugal treatment is 3500-4200 r/min, and the centrifugal time is 6-10 minutes; the extraction amount of acetonitrile was 6ml, and the addition amount of PSA was 0.2 g; the rotating speed of the second centrifugal treatment is 3600-4500 r/min, and the centrifugal time is 6-12 minutes; the extraction amount of the supernatant is 5 ml; the temperature when nitrogen is blown is 45-55 ℃.
7. The method for rapidly detecting the acrylamide content in tea leaves according to claim 6, wherein the method comprises the following steps: the optimal conditions of the first centrifugal treatment in the step S2 are that the rotating speed is 4000r/min and the centrifugal time is 8 minutes; the optimal conditions of the second centrifugal treatment are that the rotating speed is 4000r/min and the centrifugal time is 8 minutes.
8. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: in the step S2, the amount of distilled water added during reconstitution was 1ml, and the filtration membrane during filtration was a 0.22 μm aqueous filtration membrane.
9. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: the conditions of the high performance liquid chromatography in the step S3 are as follows: venusil MP C18 chromatography column (4.6mm x 250mm, 5 μm,
) (ii) a Sample introduction amount: 20 mu l of the mixture; the flow rate is 0.6 mL/min; mobile phase: methanol-water (5: 95 by volume); time: for 10 min.
10. The method for rapidly detecting the acrylamide content in tea leaves according to claim 1, wherein the method comprises the following steps: the mass spectrum conditions in step S3 are: the ionization mode is ESI+A mode; the ion source temperature is 100 ℃; the desolventizing temperature is 300 ℃; gas (N)2) Flow rate: the desolventizing gas is 350L/h, and the taper hole gas is 50L/h; capillary voltage: 3.0 KV; taper hole voltage: 20V, and (3); SIR selection ion mode; the acrylamide quantitative ion m/z is 72 [, ]13C3]The acrylamide isotopically quantified ion m/z is 75. .
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