Method for simultaneously detecting acrylamide, 4-methylimidazole and 11 kinds of heterocyclic amines in tea and application thereof

Abstract

The application belongs to the technical field of detection, and particularly relates to a method for simultaneously detecting acrylamide, 4-methylimidazole and 11 kinds of heterocyclic amines in tea and application thereof. Sample extraction is performed to obtain a to-be-purified liquid; sample purification is performed by using a mixed cation exchange solid phase extraction column for split purification; UPLC-MS / MS determination is performed; results calculation and expression are performed; judgment is performed; a standard working curve is drawn, and the heterocyclic amines in the sample are quantitatively determined; the quantitative and qualitative ion pairs exist simultaneously, and the signal-to-noise ratio is greater than or equal to 3; the retention time of the chromatographic peak of the target substance in the sample solution is consistent with that of the matrix matching standard solution, and the allowable deviation is less than + / -0.5%, so it can be determined that the target substance is contained. The application effectively purifies the co-extracted substances in tea which interfere with the detection of the target substances, is beneficial to efficiently complete the detection, has high extraction efficiency, excellent purification effect, and is excellent in recovery rate, detection limit and precision.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for simultaneously detecting acrylamide, 4-methylimidazole and 11 heterocyclic amines in tea and its application. Background Technology

[0002] As one of the world's most popular beverages, tea produces byproducts during its heat processing (such as fixation, roasting, and drying). Thermo-induced Endogenous Contaminants, Typical TECs include acrylamide, methylimidazoles, and heterocyclic amines (HAAs). Studies have shown that these contaminants have potential carcinogenicity, mutagenicity, and neurotoxicity, and there may be potential synergistic toxicity effects between different TECs and caffeine. The tea matrix is ​​extremely complex, rich in interfering substances such as alkaloids, tea polyphenols, and pigments. Even more challenging is the significant difference in the physicochemical properties of these 13 target contaminants: acrylamide is a highly polar, hydrophilic, neutral small molecule; while heterocyclic amines and 4-methylimidazoles are moderately polar, basic compounds, leading to often mutually exclusive extraction and purification conditions. Therefore, existing detection methods usually only target a single category (e.g., measuring only acrylamide or only heterocyclic amines). For example, patent CN 115015443 B proposes a method for the simultaneous detection of acrylamide and methylimidazole compounds in tea and / or coffee, but there are no reports on methods for simultaneously determining the above 13 heat-processed contaminants in tea. Therefore, establishing a method capable of simultaneously detecting multiple TECs in tea is of great significance for tea quality control and risk assessment. This application proposes a novel method for the simultaneous detection of acrylamide, methylimidazoles, and heterocyclic amines. Summary of the Invention

[0003] To address the problems existing in the prior art, the purpose of this invention is to design and provide a method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea, as well as a technical solution for its application.

[0004] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea, comprising the following steps: S.1 Sample extraction: Take the tea powder sample, add internal standard solution and reagent alcohol, extract and centrifuge, collect the supernatant; add acetonitrile solution containing sodium hydroxide to the residue, add anhydrous magnesium sulfate, vortex and centrifuge, collect the supernatant and combine them, adjust the pH value to acidic, and obtain the solution to be purified; S.2 Sample purification: Split purification was performed using a hybrid cation exchange solid-phase extraction column; S.3UPLC-MS / MS determination: matrix-matched standard solutions and sample solutions were injected under ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) conditions; S.4 Calculation and Presentation of Results: The mass concentrations of the mass spectrometry parameters of the 11 heterocyclic amines in the sample were calculated using the external standard method; the mass concentrations of acrylamide and 4-methylimidazole in the sample were calculated using the internal standard method. S.5 Judgment: Plot a 5-point external standard curve with mass concentration X as the abscissa and peak area ratio Y as the ordinate, and quantitatively determine heterocyclic amines; when quantitative and qualitative ion pairs are present at the same time, and the signal-to-noise ratio is ≥3, and the retention time of the chromatographic peak of the analyte in the sample solution deviates from that of the matrix-matched standard solution by less than ±0.5%, it can be determined that the sample contains heterocyclic amines. Acrylamide and 4-methylimidazole are determined by quantifying the peak area ratio of the target analyte to the internal standard in the sample and the additive sample. When the target analyte and the internal standard are present in both quantitative and qualitative ion pairs, and the signal-to-noise ratio is ≥3, and the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix-matched standard solution with an allowable deviation of less than ±0.5%, then acrylamide and 4-methylimidazole can be identified.

[0005] Furthermore, S.1 specifically includes the following steps: weighing tea samples, adding internal standard solution, and allowing to stand for equilibrium; then adding reagent alcohol, extracting by sonication, centrifuging, and collecting the supernatant; adding acetonitrile solution of NaOH aqueous solution to the residue, sonicating, adding anhydrous magnesium sulfate, vortexing, centrifuging, and collecting the supernatant; combining the two supernatants and adjusting the pH to 3-5.

[0006] Furthermore, the reagent alcohol described in S.1 is composed of ethanol, methanol and isopropanol, with a volume ratio of ethanol, methanol and isopropanol of 10:5:5-7:5:5.

[0007] Further, after pre-washing the PCX column with methanol, water and reagent alcohol in step S.2, the solution to be purified obtained in step S.1 is loaded onto the column; neutral acrylamide flows out with the effluent, and the effluent is collected; the PCX column is rinsed with formic acid water and methanol and then eluted with alkaline methanol, and the eluent is collected; the two collected portions are evaporated to dryness and then diluted to volume with methanol and water for instrument analysis.

[0008] Further, the PCX column described in S.2 is activated sequentially with methanol, water and reagent alcohol, the liquid to be purified is loaded onto the column and the eluent is collected; the PCX column is washed sequentially with 1-3% formic acid aqueous solution and methanol, and eluted with 10-14% ammonia-methanol solution, and the eluent is collected; the two collected liquids are evaporated to dryness by rotary evaporation, reconstituted with 1 mL of 10% (V+V) methanol aqueous solution, and filtered through a membrane.

[0009] Furthermore, the S.3 ultra-high performance liquid chromatography-tandem mass spectrometry technique employs an ACQUITY UPLC HSS-T3 column for chromatographic separation; the mass spectrometry uses multiple reaction monitoring (MRM) and ESI modes. + The model analyzed 13 pollutants; The mass spectrometry parameters of the acrylamide, 4-methylimidazole, and 11 heterocyclic amines are as follows: .

[0010] Furthermore, the chromatographic column specifications in S.3 are 100 mm × 2.1 µm and 1.8 µm; Waters, Milford, MA, USA.

[0011] Furthermore, the high-performance liquid chromatography (HPLC) conditions in S.3 are as follows: the mobile phase is 0.1% formic acid aqueous solution A and methanol B; the flow rate is 0.2-0.3 mL / min; the gradient elution program is: 0–1.5 min, 98.5% A; 1.5–3.0 min, 98.5%–50% A; 3.0–6.0 min, 50%–30% A; 6.0–7.0 min, 30%–10% A; 7.01–11.0 min, 98.5% A; the column temperature is 35-45℃; and the injection volume is 4-6 μL.

[0012] Furthermore, the mass spectrometry conditions in S.3 are as follows: electrospray voltage: 3.0 kV; ion source temperature: 150℃; desolvation temperature: 350℃; desolvation gas and cone backflush gas are nitrogen, with flow rates of 700 L / hr and 60 L / hr, respectively; collision gas Ar flow rate: 0.35 mL / min.

[0013] A second aspect of the present invention provides the application of any of the methods described in tea quality control and risk assessment.

[0014] The present invention has the following beneficial effects: (1) This application uses a Cleanert PCX solid phase extraction column to purify the test sample based on a split solid phase extraction strategy, which can effectively purify the co-extracted substances in tea that interfere with the detection of the target analyte.

[0015] (2) This application adopts UPLC-MS / MS detection conditions specific to the target object, which have excellent detection conditions and are conducive to completing the detection efficiently.

[0016] (3) This application not only has high extraction efficiency and excellent purification effect, but also meets the detection requirements in terms of recovery rate, detection limit and precision. Attached Figure Description

[0017] Figure 1The total ion flow chromatograms of 13 thermal processing contaminants under optimized conditions are shown.

[0018] Figure 2 The effect of ammonia concentration in the eluent on the recovery rates of methylimidazole and 11 heterocyclic amines was investigated. Detailed Implementation

[0019] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0020] Example 1: Establishment of mass spectrometry conditions ESI + In this mode, the precursor ions of acrylamide, 4-methylimidazole, and 11 heterocyclic amines were obtained using full-scan mass spectrometry. Taking acrylamide as an example, the m / z value was 71.9. Further optimization of the cone voltage (10-70 V) yielded the highest response at 26 V. Secondary mass spectrometry fragmentation was performed on the precursor ions, and the collision fragmentation energy (10-70 eV) was optimized to obtain the characteristic fragment ions with the highest acrylamide ion abundance at m / z 54.9 and 27.0, with optimal collision energies of 6 and 12 eV. The m / z pair with the highest response (71.9 > 54.9) was selected as the quantitative ion pair, and the other pair was used as the qualitative ion pair. The same optimization method was used to obtain the qualitative and quantitative ion pairs for 4-methylimidazole and 11 heterocyclic amines. Mass spectra are attached. Figure 1 .

[0021] Example 2: Optimization of extraction solvents for acrylamide, 4-methylimidazole, and 11 heterocyclic amines The extraction effects of acetone, acetonitrile, and reagent alcohol (90% ethanol + 5% methanol + 5% isopropanol) were compared. The results showed that while the reagent alcohol was effective in extracting the target analytes, heterocyclic amines could not be completely extracted. A two-step extraction method was further explored. In the second step, reagent alcohol, acetone, and an acetonitrile solution containing 10% (v / v) 1 M NaOH were used. It was found that using the acetonitrile solution containing 10% (v / v) 1 M NaOH in the second step significantly improved the recovery rate of heterocyclic amines (from <60% to 75.72%-121.08%). This is because the alkaline environment promotes the molecular state of heterocyclic amines, making them easier to extract with organic solvents.

[0022] Table 1. Effect of extraction solvent on the recovery rates of acrylamide, 4-methylimidazole and 11 heterocyclic amines in tea (acrylamide and methylimidazole were added at a level of 0.1 mg / kg, and heterocyclic amines were added at a level of 0.01 mg / kg, n=3). .

[0023] Example 3: Determination of Acrylamide, 4-Methylimidazole and 11 Heterocyclic Amines in Tea by Cleanert TPT Column and Split-Flow Cleanert PCX Solid Phase Extraction Method The recoveries of acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea were compared using Cleanert TPT column and split-flow Cleanert PCX solid-phase extraction methods. The results are shown in Table 2. When using Cleanert TPT solid-phase extraction for purification, many heterocyclic amines were undetectable. However, when using split-flow Cleanert PCX solid-phase extraction, the recoveries of all amines in the tea matrix, except for 4-methylimidazole, Harman, and Norharman, reached over 69%. Therefore, Cleanert PCX solid-phase extraction was chosen for the pretreatment of tea.

[0024] Table 2. Effects of Cleanert TPT column and split-flow Cleanert PCX solid-phase extraction on the recovery rates of acrylamide, 4-methylimidazole and 11 heterocyclic amines in tea (acrylamide and methylimidazole were added at 0.1 mg / kg, heterocyclic amines at 0.01 mg / kg, n=3). .

[0025] Example 4: Optimization of Elution Solvent Concentration Elution was performed on a Cleanert PCX solid-phase extraction column with ammonia-methanol concentrations of 5%, 7%, 10%, 12%, and 15%. It was found that the recovery rate of heterocyclic amines increased with increasing ammonia concentration from 5% to 12%. When 12% ammonia-methanol was used for elution, the recoveries of 11 heterocyclic amines ranged from 76.19% to 98.66% (RSD < 14.06%). At a 15% ammonia concentration, the recoveries of 4,8-DiMelQx and 7,8-DiMelQx decreased to 40.66% and 36.80%, respectively. Therefore, 12% ammonia-methanol was selected as the elution solution for the Cleanert PCX solid-phase extraction column. Figure 2 Since the recovery rate of 4-methylimidazole was still low, it was corrected using its internal standard solution. After correction, the recovery rate was 107.03% with an RSD of 9.46%.

[0026] 1. Reagents and Materials 1.1 Reagents: Acetonitrile (chromatographic grade), reagent alcohol (chromatographic grade), methanol (chromatographic grade), formic acid (chromatographic grade), ammonia (analytical grade, concentration 25%-28%), hydrochloric acid (analytical grade), anhydrous magnesium sulfate (analytical grade), sodium hydroxide (analytical grade), 11 heterocyclic amine mixed standard solution (IQ, MeIQx, Norharman, Harman, 4,8-DiMeIQx, 7,8-DiMeIQx, PHIP, AaC, 4,7,8-TriMeIQx, Glu-p-2, lu-p-1): 100 μg / mL, d3-4-methylimidazole standard solution: 100 μg / mL, 4-methylimidazole standard substance: 99.0%. 13 C3-Acrylamide standard solution: 100 μg / mL, Acrylamide standard solution: 1000 μg / mL, Standard stock solution, Mixed standard solution, Mixed internal standard solution, Standard intermediate solution, Standard working solution.

[0027] Standard stock solution: Accurately weigh 0.010 g (accurate to 0.0001 g) of the standard substance into a 100 mL volumetric flask, dilute to volume with methanol to prepare a 100 mg / L stock standard solution, and store at -20 ℃.

[0028] Mixed standard solution: Accurately measure 1 mL of each 100 mg / L standard solution (accurate to 0.01 mL) into a 10 mL volumetric flask, dilute to volume with methanol to prepare a 10 mg / L stock standard solution, and store at -20 ℃. Dilute stepwise to prepare a working standard solution of appropriate concentration.

[0029] Mixed internal standard solution: Accurately measure 1 mL of each 100 mg / L internal standard solution (accurate to 0.01 mL) into a 10 mL volumetric flask, dilute to volume with methanol to prepare a 10 mg / L stock standard solution, and store at -20 ℃.

[0030] 1.2 Materials: Cleanert PCX solid-phase extraction column (200 mg / 6 mL), microporous filter membrane (0.22 μm), organic phase.

[0031] 2. Instruments and equipment High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometer: Equipped with an (ESI) ion source, analytical balance with sensitivities of 0.00001g and 0.01g, vortex mixer, solid sample grinder, centrifuge, and ultrasonic instrument.

[0032] 3. Measurement Procedure 3.1 Sample Extraction Accurately weigh 1 g of tea sample, add 100 μL of internal standard solution with a concentration of 1 mg / kg, and let stand for at least 24 hours to equilibrate; add 10 mL of reagent alcohol (volume ratio of 90% ethanol, 5% methanol and 5% isopropanol), mix well, sonicate for 20 min, centrifuge at 12000 rpm for 5 min, and collect the supernatant. Add 10 mL of acetonitrile solution containing 10% (v / v) 1 M NaOH aqueous solution to the residue, sonicate for 20 min, add anhydrous magnesium sulfate, vortex for 30 s; centrifuge at 12000 rpm for 5 min, and collect the supernatant; Combine the two supernatants, take 10 mL of the combined solution, and immediately add 1 mol / L hydrochloric acid to adjust the pH to 4, then wait for purification.

[0033] 3.2 Sample purification The Cleanert PCX column (200 mg / 6 mL) was activated sequentially with 6 mL of methanol, 6 mL of water, and 2 mL of reagent alcohol. 10 mL of the solution to be purified was loaded onto the column, and the eluent was collected. The PCX column was then eluted sequentially with 3 mL of 2% formic acid aqueous solution and 6 mL of methanol, followed by elution with 6 mL of 12% ammonia-methanol solution. The eluent was collected. Both collected solutions were rotary evaporated to dryness, reconstituted with 1 mL of 10% methanol aqueous solution, and filtered through a 0.22 μm filter membrane for instrument detection.

[0034] 3.3 Measurement Since test conditions depend on the instrument used, it is impossible to provide universal parameters for chromatographic analysis. The following operating conditions have proven suitable for the tests. Specific parameters are shown in Tables 3 and 4.

[0035] Table 3 Liquid Chromatography Parameters .

[0036] Table 4 Mass Spectrometry Conditions .

[0037] The detection limits for acrylamide and methylimidazole in this method are 0.02 mg / kg, and the detection limits for 11 heterocyclic amines are 0.005 mg / kg.

[0038] Recovery rate The recovery experiment of this method was conducted at three spiking concentrations. Under the experimental conditions determined by this method, three experiments were performed for each spiking concentration. The recovery rate and precision results are shown in Table 5.

[0039] Table 5. Linearity, concentration, and recovery range of acrylamide, 4-methylimidazole, and 11 heterocyclic amines in green tea. .

[0040] Example 5: A method for simultaneous detection of acrylamide, 4-methylimidazole and 11 heterocyclic amines in tea leaves S.1 Sample Extraction: Weigh the pulverized tea sample, add internal standard solution, and allow to stand for equilibrium; add reagent alcohol, vortex mix, ultrasonically extract, and collect the first supernatant after centrifugation; add acetonitrile solution containing sodium hydroxide to the residue for a second ultrasonic extraction, add anhydrous magnesium sulfate, vortex, centrifuge, and collect the second supernatant; combine the two supernatants and immediately adjust the pH to acidic with hydrochloric acid to obtain the purified solution.

[0041] The sample extraction in step S.1 specifically includes: weighing 1 g of tea sample, adding 100 μL of internal standard solution with a concentration of 1 mg / kg, and allowing it to stand for at least 24 hours to equilibrate; adding 10 mL of reagent alcohol (volume ratio of 90% ethanol, 5% methanol, and 5% isopropanol), mixing well, sonicating for 20 min, centrifuging at 12000 rpm for 5 min, and collecting the supernatant; adding 10 mL of acetonitrile solution containing 10% (v / v) 1 M NaOH aqueous solution to the residue, sonicating for 20 min, adding 1 g of anhydrous magnesium sulfate, vortexing for 30 s, centrifuging at 12000 rpm for 5 min, and collecting the supernatant; combining the two supernatants, taking 10 mL of the combined solution, and immediately adding 1 mol / L hydrochloric acid to adjust the pH to 4.

[0042] S.2 Sample purification: Split purification was performed using a hybrid cation exchange solid-phase extraction column. After pre-washing the PCX column with methanol, water, and reagent alcohol, the sample to be purified was loaded onto the column. Neutral acrylamide flowed out with the eluent, which was collected. The PCX column was then rinsed with formic acid and methanol, followed by elution with alkaline methanol. The eluent was collected, and both portions were evaporated to dryness before being diluted to volume with methanol and water for instrument analysis.

[0043] The sample purification in step S.2 specifically includes: activating the Cleanert PCX column (200 mg / 6 mL) sequentially with 6 mL of methanol, 6 mL of water, and 2 mL of reagent alcohol; loading 10 mL of the solution to be purified onto the column and collecting the eluent; then eluting the PCX column sequentially with 3 mL of 2% formic acid aqueous solution and 6 mL of methanol, followed by elution with 6 mL of 12% ammonia-methanol solution, and collecting the eluent; evaporating both collected solutions to dryness using a rotary evaporator, reconstituted with 1 mL of 10% methanol aqueous solution, filtering through a 0.22 μm filter membrane, and preparing for instrument detection.

[0044] S.3 UPLC-MS / MS determination: Matrix-matched standard solutions and sample solutions were injected under the set HPLC-MS / MS conditions: the HPLC was performed using an ACQUITY UPLC HSS-T3 column for chromatographic separation; the mass spectrometry was performed using multiple reaction monitoring (MRM) and ESI modes.+ The model was used to analyze 13 pollutants.

[0045] The mass spectrometry parameters of acrylamide, 4-methylimidazole, and the 11 heterocyclic amines in step S.3 are as follows: Table 6. Mass spectrometric parameters of acrylamide, 4-methylimidazole, and 11 heterocyclic amines .

[0046] In step S.3, the chromatographic column specifications are 100 mm × 2.1 µm and 1.8 µm; Waters, Milford, MA, USA.

[0047] The medium-performance liquid chromatography (HPLC) conditions in step S.3 are as follows: the mobile phase is 0.1% formic acid aqueous solution A and methanol B; the flow rate is 0.25 mL / min; the gradient elution program is as follows: 0–1.5 min, 98.5% A; 1.5–3.0 min, 98.5%–50% A; 3.0–6.0 min, 50%–30% A; 6.0–7.0 min, 30%–10% A; 7.01–11.0 min, 98.5% A; the column temperature is 40℃; and the injection volume is 5 μL.

[0048] The mass spectrometry conditions in step S.3 are as follows: electrospray voltage: 3.0 kV; ion source temperature: 150℃; desolvation temperature: 350℃; desolvation gas and cone backflush gas are nitrogen, with flow rates of 700 L / hr and 60 L / hr, respectively; collision gas Ar flow rate: 0.35 mL / min.

[0049] S.4 Blank test.

[0050] S.5 Calculation and Presentation of Results: The mass concentrations of the 11 heterocyclic amines in the sample were calculated using the external standard method (Equation 1). Blank values ​​must be subtracted from the processed results. In Equation 1: ; X i -- The residual amount of the target substance in the sample, expressed in milligrams per kilogram; C i -- The concentration of the target substance obtained from the standard curve, in milligrams per liter; V – Final volume of the sample solution, in milliliters; m – The mass of the final sample liquid, in grams; Calculate the mass concentrations of acrylamide and 4-methylimidazole in the sample using the internal standard method (Equation 2). The blank value must be subtracted from the processed results. In Equation 2: ; C – Mass fraction of the target analyte in the sample, expressed in milligrams per kilogram; A – The peak area ratio of the target analyte to the internal standard in the sample; A′──The peak area ratio of the target analyte to the internal standard in the added sample; c – The mass fraction of the target analyte in the added sample, expressed in milligrams per kilogram.

[0051] S.6 Judgment: Plot a 5-point external standard curve with mass concentration X as the abscissa and peak area ratio Y as the ordinate. Use the standard curve to quantitatively determine heterocyclic amines in the sample. If the response value of the analyte in the sample solution meets the linear range of the instrument detection, and both quantitative and qualitative ion pairs are present with a signal-to-noise ratio ≥3, and the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix-matched standard solution with an allowable deviation of less than ±0.5%, then the analyte can be identified as being present.

[0052] Example 6: Application Case Green tea samples were purchased from a local market, and the contents of acrylamide, methylimidazole, and 11 heterocyclic amines were determined according to the method of this invention. 4-Methylimidazole, acrylamide, Harman, and Norharman were detected, with detection rates of 100%, 0%, 30%, and 40%, respectively. The detected sample contents ranged from 0.048 mg / kg to 0.152 mg / kg, 0.005 mg / kg to 0.009 mg / kg, and 0.006 to 0.012 mg / kg, respectively.

[0053] The above description is a preferred embodiment of the present invention and is only used to explain the present invention. It is not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea, characterized in that, Includes the following steps: S.1 Sample extraction: Take the tea powder sample, add internal standard solution and reagent alcohol, extract and centrifuge, collect the supernatant; add acetonitrile solution containing sodium hydroxide to the residue, add anhydrous magnesium sulfate, vortex and centrifuge, collect the supernatant and combine them, adjust the pH value to acidic, and obtain the solution to be purified; S.2 Sample purification: Split purification was performed using a hybrid cation exchange solid-phase extraction column; S.3UPLC-MS / MS determination: matrix-matched standard solutions and sample solutions were injected under ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) conditions; S.4 Calculation and Presentation of Results: The mass concentrations of the mass spectrometry parameters of the 11 heterocyclic amines in the sample were calculated using the external standard method; the mass concentrations of acrylamide and 4-methylimidazole in the sample were calculated using the internal standard method. S.5 Judgment: Plot a 5-point external standard curve with mass concentration X as the abscissa and peak area ratio Y as the ordinate, and quantitatively determine heterocyclic amines; when quantitative and qualitative ion pairs are present at the same time, and the signal-to-noise ratio is ≥3, and the retention time of the chromatographic peak of the analyte in the sample solution deviates from that of the matrix-matched standard solution by less than ±0.5%, it can be determined that the sample contains heterocyclic amines. Acrylamide and 4-methylimidazole were determined based on the peak area ratio of the target analyte to the internal standard in the sample and the additive sample. When the target analyte and internal standard quantitative and qualitative ion pairs are present simultaneously, and the signal-to-noise ratio is ≥3, and the retention time of the chromatographic peak of the analyte in the sample solution is consistent with that of the matrix-matched standard solution, with an allowable deviation of less than ±0.5%, then it can be determined that acrylamide and 4-methylimidazole are present.

2. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 1, characterized in that, S.1 specifically The steps include: weighing tea samples, adding internal standard solution, and allowing to stand for equilibrium; adding reagent alcohol, extracting by sonication, centrifuging, and collecting the supernatant; adding acetonitrile solution of NaOH aqueous solution to the residue, sonicating, adding anhydrous magnesium sulfate, vortexing, centrifuging, and collecting the supernatant; combining the two supernatants and adjusting the pH to 3-5.

3. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 2, characterized in that, The reagent alcohol described in S.1 is composed of ethanol, methanol and isopropanol, with a volume ratio of ethanol, methanol and isopropanol of 10:5:5-7:5:

5.

4. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 1, characterized in that, After pre-rinsing the PCX column with methanol, water, and reagent alcohol in step S.2, the purified solution obtained in step S.1 is loaded onto the column; neutral acrylamide flows out with the eluent, and the eluent is collected; the PCX column is rinsed with formic acid water and methanol, and then eluted with alkaline methanol, and the eluent is collected; the two collected portions are evaporated to dryness and then diluted to volume with methanol and water for instrument analysis.

5. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 4, characterized in that, S.2 The PCX column was activated sequentially with methanol, water and reagent alcohol. The solution to be purified was loaded onto the column and the eluent was collected. The PCX column was washed sequentially with 1-3% formic acid aqueous solution and methanol, and eluted with 10-14% ammonia-methanol solution. The eluent was collected. The two collected solutions were evaporated to dryness by rotary evaporation, reconstituted with 1 mL of 10% (V+V) methanol aqueous solution, and filtered through a membrane.

6. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 1, characterized in that, The S.3 ultra-high performance liquid chromatography-tandem mass spectrometry technique uses an ACQUITY UPLC HSS-T3 column for chromatographic separation; mass spectrometry employs multiple reaction monitoring (MRM) and ESI modes. + The model analyzed 13 pollutants; The mass spectrometry parameters of the acrylamide, 4-methylimidazole, and 11 heterocyclic amines are as follows: 。 7. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 6, characterized in that, The column specifications in S.3 are 100 mm × 2.1 µm and 1.8 µm; Waters, Milford, MA, USA.

8. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 7, characterized in that, The high-performance liquid chromatography (HPLC) conditions in S.3 are as follows: the mobile phase is 0.1% formic acid aqueous solution A and methanol B; the flow rate is 0.2-0.3 mL / min; the gradient elution program is: 0–1.5 min, 98.5% A; 1.5–3.0 min, 98.5%–50% A; 3.0–6.0 min, 50%–30% A; 6.0–7.0 min, 30%–10% A; 7.01–11.0 min, 98.5% A; the column temperature is 35-45℃; and the injection volume is 4-6 μL.

9. The method for simultaneously detecting acrylamide, 4-methylimidazole, and 11 heterocyclic amines in tea as described in claim 7, characterized in that, The mass spectrometry conditions in S.3 are as follows: electrospray voltage: 2.5-3.5 kV; ion source temperature: 140-160℃; desolventizing temperature: 330-370℃; desolventizing gas and cone backflush gas are nitrogen, with flow rates of 650-750 L / hr and 50-70 L / hr, respectively. Ar flow rate of the collision gas; 0.3-0.4 mL / min.

10. The application of the method as described in any one of claims 1-9 in tea quality control and risk assessment.

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