Method for detecting acrylamide content in fried food

Abstract

The application discloses a kind of detection methods of acrylamide content in fried food, it is related to food pollutant detection technical field, including the preparation extract of the to be measured fried food by homogenization treatment, by liquid-liquid extraction obtains sample extraction liquid, after adding stable isotope internal standard, derivative treatment is carried out, derivative sample is separated by gas chromatography preset temperature program, is imported into tandem mass spectrometer in multiple reaction monitoring mode and acquires acrylamide derivative compound characteristic ion pair signal, calculate relative response intensity by internal standard calibration, combined with standard curve obtains acrylamide content in sample.The method can offset the matrix interference and efficiency fluctuation in the process of pretreatment, eliminate non-target ion signal interference, strengthen the specificity of detection signal, improve the accuracy and data reliability of acrylamide quantitative detection.

Description

Technical Field

[0001] This invention belongs to the field of food contaminant detection technology, specifically a method for detecting acrylamide content in fried foods. Background Technology

[0002] Current methods for detecting acrylamide content in fried foods typically involve homogenizing the sample, performing liquid-liquid extraction, directly derivatizing the extract, separating by gas chromatography, and then acquiring the signal using conventional mass spectrometry. Quantitative calculations often rely on the external standard method, without adding a stable isotope internal standard for calibration after extraction and before derivatization. In this approach, sample matrix interference, component loss during extraction, and differences in derivatization efficiency all directly affect the signal output. The external standard quantification method cannot synchronously correct for the influence of variables throughout the entire pretreatment process, leading to systematic shifts in the mass spectrometry response data.

[0003] Current mass spectrometry acquisition methods lack the ability to specifically screen ion pairs of target derived compounds. Ion signals from non-target matrix components can superimpose with the target analyte signal, resulting in a large amount of interference in the acquired mass spectrometry data. Furthermore, the lack of specific signal acquisition via tandem mass spectrometry in multiple reaction monitoring mode after gas chromatography separation leads to insufficient signal identification of the target analyte and unreliable raw data for quantitative calculations.

[0004] This invention addresses the quantitative bias caused by the lack of stable isotope internal standard calibration in existing detection methods, as well as the signal interference caused by non-specific mass spectrometry acquisition. It optimizes the detection process and mass spectrometry acquisition mode by adding internal standard in sequence and using dedicated ion pair acquisition methods to eliminate the influence of preprocessing variables and non-target signals on the detection results. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art; Therefore, this invention proposes a method for detecting acrylamide content in fried foods, comprising: A homogenization solution was prepared from the fried food sample to form the extract to be tested; Liquid-liquid extraction was performed on the extract to be tested to separate the extract phase containing the target analyte, resulting in a sample extract. A stable isotope internal standard is added to the sample extract to form an internally standardized sample extract. The extract of the internal standard sample is subjected to derivatization treatment to convert the target analyte into a derivatized compound with specific volatility, thereby generating a derivatized sample; The derivatized sample is injected into a gas chromatography system, and the components are separated based on a preset gas chromatography temperature program to obtain a gas chromatography elution signal. The gas chromatographic elution signal is imported into a tandem mass spectrometer, and ion pair signals corresponding to the characteristics of acrylamide-derived compounds are collected in multiple reaction monitoring mode to form mass spectrometry response data. Based on the known concentration of the stable isotope internal standard added to the internal standard sample extract, the mass spectrometry response data is calibrated using the internal standard, and the relative response intensity of the target analyte is calculated. Based on a pre-established standard curve, the relative response intensity is converted into the acrylamide content value in the fried food sample to be tested.

[0006] Further, liquid-liquid extraction is performed on the extract to be tested to separate the extract phase containing the target analyte, resulting in a sample extract, comprising: The extract to be tested was transferred to a separatory container and a non-polar organic extraction solvent was added. The extract to be tested containing a non-polar organic extraction solvent was shaken and mixed, and then allowed to stand to separate into layers, forming a liquid-liquid system consisting of an upper aqueous phase and a lower organic phase. Collect the lower organic phase, which is an extract phase containing acrylamide; The collected extract phase was concentrated by rotary evaporation to remove some of the organic solvent, resulting in a concentrated sample extract.

[0007] Furthermore, prior to the rotary evaporation concentration of the collected extract phase, the process further includes: The lower organic phase is passed through a chromatography column packed with a desiccant to remove residual moisture, resulting in a dry extract phase. The rotary evaporation concentration process is performed on the dried extract phase.

[0008] Further, a stable isotope internal standard is added to the sample extract to form an internally standardized sample extract, comprising: The stable isotope internal standard is an acrylamide solution labeled with carbon-13 isotope; Accurately transfer a fixed volume of carbon-13 isotope-labeled acrylamide stock solution using a micropipette; The carbon-13 isotope-labeled acrylamide stock solution was injected into the sample extract to obtain a mixed solution; The mixed solution is vortexed to ensure that the carbon-13 isotope-labeled acrylamide is evenly distributed in the mixed solution, thereby forming the internal standard sample extract.

[0009] Further, the extract of the internal standard sample is derivatized to convert the target analyte into a derivatized compound with specific volatility, generating a derivatized sample, including: Add trifluoroacetic anhydride derivatization reagent to the extract of the internally standardized sample; The extract of the internal standard sample after adding trifluoroacetic anhydride derivatization reagent was heated in a constant temperature water bath to promote the reaction of acrylamide with the trifluoroacetic anhydride derivatization reagent to generate a trifluoroacetic acid derivative of acrylamide. The reaction mixture after water bath heating was subjected to nitrogen blowing to remove excess derivatization reagents and byproducts, resulting in the derivatized sample with the trifluoroacetate derivative of acrylamide as the main analytical target.

[0010] Further, the derivatized sample is injected into a gas chromatography system, and component separation is performed based on a preset gas chromatography temperature program to obtain a gas chromatography elution signal, including: The derivatized sample was injected into the column from the injection port of the gas chromatography system using a splitless injection mode. The preset gas chromatography temperature program is executed, which includes the setting of initial column oven temperature, programmed temperature ramp rate and final column oven temperature. During the operation of the preset gas chromatography temperature program, the components in the derivatized sample migrate at different rates in the chromatographic column due to differences in volatility, and are eventually separated and eluted sequentially. At the end of the chromatographic column, a detector records the effluent signal that changes over time; the effluent signal is the gas chromatographic effluent signal.

[0011] Further, the gas chromatographic elution signal is imported into a tandem mass spectrometer, and ion pair signals corresponding to the characteristics of acrylamide-derived compounds are acquired in multiple reaction monitoring mode to form mass spectrometry response data, including: The gas chromatographic elution signal is directly imported into the ion source of the tandem mass spectrometer via an interface, and the ion source performs electron bombardment ionization on the elution. In the first-stage mass analyzer of the tandem mass spectrometer, the parent ion corresponding to the trifluoroacetate derivative of the acrylamide is selected; The selected parent ion is introduced into the collision cell and undergoes cleavage upon collision with the inert gas to produce daughter ions; In the second-stage mass analyzer of the tandem mass spectrometer, pre-defined characteristic ions related to the structural features of acrylamide-derived compounds are screened. The curves of the signal intensity of the ion pair composed of the parent ion and the characteristic daughter ion changing over time are recorded, and the curves constitute the mass spectrometry response data.

[0012] Further, based on the known concentration of the stable isotope internal standard added to the internally standardized sample extract, the mass spectrometry response data is calibrated using an internal standard, and the relative response intensity of the target analyte is calculated, including: From the mass spectrometry response data, the signal peak area of ​​the characteristic daughter ion corresponding to the acrylamide derivative compound is extracted and recorded as the target peak area; From the mass spectrometry response data, the signal peak area of ​​the characteristic daughter ion corresponding to the carbon-13 isotope-labeled acrylamide derivative compound is extracted and recorded as the target internal standard peak area; The relative response intensity is obtained by calculating the ratio of the peak area of ​​the target substance to the peak area of ​​the target internal standard. The relative response intensity eliminates the influence of injection volume fluctuation and instrument response drift.

[0013] Further, based on a pre-established standard curve, the relative response intensity is converted into a numerical value of acrylamide content in the fried food sample to be tested, including: Obtain the pre-established standard curve, which is a calibration curve plotted with the relative response intensity of acrylamide standard solutions of different concentrations as the ordinate and their corresponding concentrations as the abscissa. Substitute the relative response intensity corresponding to the acrylamide derivative compound in the fried food sample to be tested into the mathematical relationship of the standard curve; Solve the mathematical relationship to calculate the concentration of acrylamide in the extract of the internally standardized sample; By combining the sample size of the fried food sample to be tested and the final volume of the sample extract, the calculated concentration of acrylamide in the internal standard sample extract is converted into the content of acrylamide in the fried food sample to be tested.

[0014] Furthermore, the process of establishing the pre-established standard curve includes: Prepare a series of acrylamide standard solutions with different known concentrations; A fixed amount of the stable isotope internal standard is added to each of the acrylamide standard solutions to form a series of internally standardized standard solutions. For each internal standard solution in the series of internal standard solutions, the same derivatization, gas chromatography separation and tandem mass spectrometry detection process as that of the internal standard sample extract is performed. Record the mass spectrometry response of each internal standard solution in the series of internal standard solutions, and calculate the relative response intensity of each internal standard solution; Using the concentration of the acrylamide standard solution as the abscissa and its corresponding relative response intensity as the ordinate, a linear regression fitting was performed to obtain the standard curve and its mathematical relationship.

[0015] Compared with the prior art, the beneficial effects of the present invention are: A stable isotope internal standard is added to the sample extract obtained from liquid-liquid extraction to form an internally standardized sample extract. The mass spectrometry response data is then calibrated using the known concentration of the stable isotope internal standard, and the relative response intensity of the target analyte is calculated. Matrix interference, extraction loss, and differences in derivatization reaction efficiency during sample pretreatment can be simultaneously offset, narrowing the fluctuation range of the mass spectrometry response data. The correlation between the relative response intensity and the actual content of the target analyte is closer, and the content values ​​after standard curve conversion closely match the true composition of the sample. The influence of variables in the pretreatment stage can be simultaneously weakened, and the accuracy of the quantitative calculation is optimized.

[0016] After separation by gas chromatography with a preset temperature program, the derivatized sample is introduced into a tandem mass spectrometer. In multiple reaction monitoring mode, characteristic ion pair signals corresponding to the acrylamide derivatized compounds are acquired to form specific mass spectrometry response data. Interfering ion signals from non-target components can be effectively eliminated, retaining only the ion pair response information matching the target derivatized compound. This enhances the signal specificity of mass spectrometry detection, reduces the interference of matrix components on the detection signal, further reduces error sources in the content value conversion process, and makes the correspondence between the detection signal and the target analyte more pure, thus improving the specificity of the mass spectrometry response data. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the steps of a method for detecting acrylamide content in fried foods according to the present invention. Figure 2 A flowchart for adding internal standards for stabilizing isotopes; Figure 3 Radar chart for comprehensive performance evaluation of acrylamide derivatization process; Figure 4 Line graph of the temperature program for a gas chromatography column oven; Figure 5 A curve showing the peak area comparison between the target analyte and the internal standard. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] See Figure 1This invention provides a method for detecting acrylamide content in fried foods. The method includes: preparing a homogenized solution from a sample of fried food to form an extract to be tested; performing liquid-liquid extraction on the extract to separate the extract phase containing the target analyte, obtaining a sample extract; adding a stable isotope internal standard to the sample extract to form an internally standardized sample extract; subjecting the internally standardized sample extract to derivatization to convert the target analyte into a derivatized compound with specific volatility, generating a derivatized sample; injecting the derivatized sample into a gas chromatography system, performing component separation based on a preset gas chromatography temperature program, and obtaining a gas chromatography elution signal; importing the gas chromatography elution signal into a tandem mass spectrometer, and acquiring ion pair signals corresponding to the characteristics of the acrylamide derivatized compound in multiple reaction monitoring mode to form mass spectrometry response data; and performing internal standard calibration on the mass spectrometry response data based on the known concentration of the stable isotope internal standard added to the internally standardized sample extract to calculate the relative response intensity of the target analyte. Based on a pre-established standard curve, the relative response intensity is converted into the acrylamide content value in the fried food sample to be tested.

[0020] In one embodiment of the present invention, liquid-liquid extraction is performed on the analyte to separate the extract phase containing the target analyte, obtaining a sample extract. This includes the following steps: transferring the analyte to a separatory container and adding a non-polar organic extraction solvent. The analyte containing the non-polar organic extraction solvent is shaken and mixed, then allowed to separate into layers, forming a liquid-liquid system comprising an upper aqueous phase and a lower organic phase. The lower organic phase, which is an extract phase containing acrylamide, is collected. Before performing rotary evaporation concentration on the collected extract phase, the lower organic phase is passed through a chromatography column packed with a desiccant to remove residual water, obtaining a dried extract phase. The collected dried extract phase is then subjected to rotary evaporation concentration to evaporate and remove some of the organic solvent, obtaining the concentrated sample extract.

[0021] In practice, the process of obtaining the sample extract through liquid-liquid extraction is performed. Using fried potato chips as an example, 5.0 g of uniformly pulverized potato chip sample is weighed and 20 mL of ultrapure water is added for vortexing and ultrasonic extraction. The sample is then centrifuged, and the supernatant is collected; this supernatant is the extract to be tested. All of the extract is transferred to a 50 mL polytetrafluoroethylene separatory funnel, and 15 mL of chromatographically pure ethyl acetate is added as a non-polar organic extraction solvent. The separatory funnel is manually shaken for approximately 1 minute, and then placed on an iron stand to stand for 10 minutes until the mixture completely separates into two distinct liquid-liquid phases, forming a clear liquid-liquid system containing an upper aqueous phase and a lower organic phase. The stopcock of the separatory funnel is opened, and the entire lower organic phase is slowly released and collected; this lower organic phase is the extract containing acrylamide.

[0022] In some embodiments, the nonpolar organic extraction solvent may also be methyl tert-butyl ether or dichloromethane, with the volume ratio of the added solvent to the volume of the extract to be tested ranging from 0.5:1 to 2:1. In some embodiments, the extract to be tested containing the nonpolar organic extraction solvent may be mixed by shaking using a mechanical shaker, with the shaking speed set at 200 rpm and the shaking time at 5 minutes. The settling time for phase separation may be adjusted depending on the solvent system to ensure complete separation of the two phases. Optionally, before the collected extract phase is concentrated by rotary evaporation, the lower organic phase is passed through a glass chromatography column packed with 5 g of anhydrous sodium sulfate. The anhydrous sodium sulfate is pre-activated at 105°C for 2 hours, and the bottom of the chromatography column is packed with a small amount of glass wool. The lower organic phase is passed through the chromatography column at a flow rate of approximately 1 drop per second, and the eluent organic phase is collected to obtain a dried extract phase. It is understood that this drying step aims to remove any trace amounts of moisture that may be entrained in the lower organic phase; the use of anhydrous sodium sulfate as a drying agent is a common choice, but it can also be replaced with anhydrous magnesium sulfate. The collected, dried extract phase was concentrated by rotary evaporation, and then transferred to a 50 mL round-bottom flask. The flask was then mounted in a rotary evaporator, the water bath temperature was set to 35°C, the system pressure was adjusted to 200 mbar, and the rotary evaporator was started. The evaporation process continued until the liquid volume in the round-bottom flask was reduced to approximately 0.5 mL, at which point the concentrated sample extract was obtained. It is understood that the purpose of rotary evaporation is to remove most of the organic solvent to achieve enrichment. The concentrated sample extract needs to be transferred to a vial, and the round-bottom flask is washed with a small amount of organic solvent and brought to a final volume of 1.0 mL for subsequent analysis. In practice, to compare the effect of the drying step, two identical extracts were processed in parallel: one was directly concentrated by rotary evaporation after extraction, while the other underwent a drying step before rotary evaporation. Observations revealed that samples without the drying step sometimes showed tiny water droplets at the bottom of the flask after rotary evaporation, and the uniformity of the concentrate was slightly poor during transfer and volume adjustment; while the concentrates of samples that underwent the drying step were homogeneous organic phases, which were easy to transfer and adjust to volume.

[0023] In one embodiment of the present invention, a stable isotope internal standard is added to the sample extract to form an internally standardized sample extract. (See also...) Figure 2 The stable isotopic internal standard is a carbon-13 isotope-labeled acrylamide solution. A fixed volume of the carbon-13 isotope-labeled acrylamide stock solution is accurately transferred using a micropipette and injected into the sample extract to obtain a mixed solution. The mixed solution is vortexed to ensure uniform distribution of the carbon-13 isotope-labeled acrylamide, thereby forming the internal standard sample extract.

[0024] In the specific implementation, the process of constructing the internal standard sample extract is illustrated using the concentrated sample extract obtained in the previous example. Assuming that after rotary evaporation concentration and re-volume adjustment, the resulting sample extract volume is 1.0 mL, it is transferred to a 2 mL liquid chromatography vial. The stable isotope internal standard is a carbon-13 isotope-labeled acrylamide solution, with a stock concentration calibrated to 1.0 μg / mL. Using a calibrated 10-100 μL micropipette, with the pipette range adjusted to 20.0 μL and a new, non-contamination-free tip attached, 20.0 μL of the carbon-13 isotope-labeled acrylamide stock solution is accurately transferred. The pipette tip is immersed below the surface of the sample extract, and the transferred 20.0 μL of the carbon-13 isotope-labeled acrylamide stock solution is slowly injected into the sample extract to obtain a mixed solution.

[0025] In some embodiments, the concentration of the carbon-13 isotope-labeled acrylamide stock solution can be adjusted according to the estimated acrylamide content in the sample to be tested, for example, using a stock solution of 0.1 μg / mL or 5.0 μg / mL, with a transfer volume selected between 5.0 μL and 50.0 μL, aiming to make the amount of internal standard added close to the amount of the target analyte in the sample. In some embodiments, to evaluate the repeatability of the pipetting operation, the internal standard can be added three times in parallel to the same sample extract, with 20.0 μL of internal standard stock solution transferred to three equal aliquots of the same sample extract for each of the three operations. Optionally, after injecting the transferred carbon-13 isotope-labeled acrylamide stock solution into the sample extract, it can be gently mixed several times by pipetting before the injection vial is tightly capped. The tightly capped sample vial was vortexed. The vial was placed on a vortex mixer, and the oscillation speed was set to 2500 rpm for 30 seconds to ensure uniform distribution of the carbon-13 isotope-labeled acrylamide in the mixed solution, thus forming the internal standard sample extract. It is understood that vortexing is an effective means of achieving rapid and uniform mixing. Insufficient oscillation time may lead to uneven local concentrations of the internal standard in the solution, while excessive oscillation time offers no additional benefit and may cause solvent evaporation.

[0026] In practical implementation, to illustrate the precise calculation of the added internal standard and its significance in subsequent quantification, the following relationship is introduced: Absolute amount of added internal standard The concentration of the transferred internal standard stock solution and transfer volume The decision is: in: The absolute mass of the added C13-labeled acrylamide is expressed in nanograms. The concentration of carbon-13 isotope-labeled acrylamide stock solution is expressed in micrograms per milliliter. This indicates the volume of the stock solution transferred, in microliters. Calculated using the example data above, It is understandable that this absolute amount is a precisely known constant used to calibrate the target analyte's response after mass spectrometry detection. To compare the effect of different added volumes on the results, a comparative experiment can be designed: prepare two equal volumes of sample extract from the same sample, thoroughly mixed, one with 20.0 μL of internal standard stock solution added, and the other with 10.0 μL of internal standard stock solution added. Under identical subsequent derivatization and detection conditions, the final calculated peak area ratio of the target analyte to the internal standard will show a systematic difference due to the different amounts of internal standard added. However, through conversion using a pre-established standard curve, the acrylamide content values ​​calculated for the two samples should ideally be consistent. This verifies that the internal standard method can correct for variations in pretreatment and injection processes in quantification.

[0027] In one embodiment of the present invention, the internal standard sample extract is derivatized to convert the target analyte into a derivatized compound with specific volatility, generating a derivatized sample. A trifluoroacetic anhydride derivatizing reagent is added to the internal standard sample extract, and the extract is then heated in a constant-temperature water bath to promote the reaction between acrylamide and the trifluoroacetic anhydride derivatizing reagent, generating a trifluoroacetic acid derivative of acrylamide. The reaction mixture after water bath heating is then subjected to nitrogen blowing to remove excess derivatizing reagent and byproducts, yielding the derivatized sample with the trifluoroacetic acid derivative of acrylamide as the primary analyte.

[0028] In specific implementation, the process of generating the derivatized sample is illustrated using the internal standard sample extract obtained in the previous example. Assume the volume of the internal standard sample extract is 1.02 mL, placed in a 2 mL threaded vial. Add 50 μL of trifluoroacetic anhydride derivatization reagent to the internal standard sample extract. Use a microsyringe to draw the trifluoroacetic anhydride derivatization reagent, insert the needle below the surface of the internal standard sample extract, and slowly push it in to complete the addition operation. In some embodiments, the amount of trifluoroacetic anhydride derivatization reagent added can be adjusted according to the volume of the reaction system, ranging from 20 μL to 100 μL. In some embodiments, to prevent violent hydrolysis of trifluoroacetic anhydride upon contact with water, the addition operation can be performed quickly in a glove box or under a dry nitrogen atmosphere. After addition, immediately tighten the vial cap with a polytetrafluoroethylene liner. Optionally, after adding the trifluoroacetic anhydride derivatization reagent, the vial can be gently shaken manually for a few seconds for preliminary mixing. The internal standard sample extract, after adding the trifluoroacetic anhydride derivatizing reagent, was heated in a constant-temperature water bath. The vial was placed in a metal heating block or water bath. The heating temperature was set to 70 degrees Celsius, and the heating time was 30 minutes to promote the reaction between acrylamide and the trifluoroacetic anhydride derivatizing reagent, generating a trifluoroacetic acid derivative of acrylamide. It is understood that constant-temperature water bath heating provides the necessary energy for the derivatization reaction, and temperature and time are the main factors affecting the degree of reaction completion. To evaluate the effect of different water bath conditions, a comparative experiment can be designed, using different combinations of heating temperatures and times to treat the same batch of internal standard sample extracts. After the reaction, subsequent nitrogen blowing and detection were performed uniformly. The mass spectrometry response peak areas of the acrylamide trifluoroacetic acid derivative generated under different conditions differ. The relative reaction efficiency can be evaluated using the following relationship: Relative conversion rate of derivatization reaction: in: It represents the relative conversion rate of a derivatization reaction and is a dimensionless ratio. This represents the peak area of ​​characteristic ions of acrylamide derivatives detected under specific water bath conditions. This represents the peak area of ​​characteristic ions of acrylamide derivatives detected under the selected reference water bath conditions. See Table 1 for comparative data.

[0029] Table 1: Effects of different water bath conditions on the mass spectrometry response of acrylamide derivatives In practice, after the constant temperature water bath heating is completed, the sample vial is removed from the heating device and cooled at room temperature for about 5 minutes. The reaction mixture after water bath heating is then subjected to nitrogen purging. The vial cap is opened, and the vial opening is aligned with the purging needle of the nitrogen purging apparatus. The nitrogen pressure is adjusted to create stable bubbles on the liquid surface, and the purging temperature is set to 40 degrees Celsius. Nitrogen purging continues until the volume of the reaction mixture is dried and concentrated to approximately 50 microliters. At this point, excess trifluoroacetic anhydride derivatizing reagent and volatile byproducts have been removed, yielding the derivatized sample with the trifluoroacetic acid derivative of acrylamide as the primary analytical target. It is understood that nitrogen purging utilizes inert nitrogen to remove volatile reagents, and the purging temperature needs to be higher than the solvent boiling point to accelerate evaporation, but lower than the decomposition temperature of the target derivative. Optionally, near the end of the nitrogen blowing process, 50 μL of ethyl acetate can be added to the residue at the bottom of the vial for redissolution, and then briefly vortexed again to ensure that the derivatized sample is completely dissolved and homogeneous, facilitating subsequent instrument analysis.

[0030] See Figure 3 This is a radar chart evaluating the comprehensive performance of the acrylamide derivatization process, used to assess the optimal conditions for its application in fried foods. The reaction conversion rate represents the efficiency of acrylamide conversion to the derivative under a 70℃ / 30min water bath condition; product stability reflects the stability of the derivative during pretreatment and storage, showing good performance; peak area intensity corresponds to the signal intensity of characteristic ions in the derivative during mass spectrometry detection, directly affecting detection sensitivity; reproducibility indicates the consistency of results from multiple parallel experiments, reflecting process reliability; time efficiency combines reaction time and detection efficiency, and under the premise of ensuring conversion rate, a 30min water bath is the optimal duration. The chart visually demonstrates the balance of each indicator, allowing for quick identification of any shortcomings in the current process and clearly presenting the comprehensive performance of the derivatization process.

[0031] In one embodiment of the present invention, the derivatized sample is injected into a gas chromatography system, and component separation is performed based on a preset gas chromatography temperature program to obtain a gas chromatography elution signal. Using a splitless injection mode, the derivatized sample is injected into the chromatographic column from the injection port of the gas chromatography system, and the preset gas chromatography temperature program is executed. The temperature program includes settings for an initial column oven temperature, a programmed temperature ramp rate, and a final column oven temperature. During the operation of the preset gas chromatography temperature program, the components in the derivatized sample migrate at different rates in the chromatographic column due to differences in volatility, eventually separating and eluting sequentially. At the end of the chromatographic column, a detector records the elution signal changing over time; this elution signal is the gas chromatography elution signal. The gas chromatography elution signal is imported into a tandem mass spectrometer, and ion pair signals corresponding to the characteristics of the acrylamide derivatized compound are acquired in multiple reaction monitoring mode to form mass spectrometry response data. The gas chromatography elution signal is directly imported into the ion source of the tandem mass spectrometer via an interface, and the ion source performs electron bombardment ionization on the elution. In the first-stage mass analyzer of the tandem mass spectrometer, the precursor ion corresponding to the trifluoroacetate derivative of acrylamide is selected and introduced into the collision cell, where it undergoes fragmentation upon collision with an inert gas, generating daughter ions. In the second-stage mass analyzer of the tandem mass spectrometer, preset characteristic daughter ions related to the structural features of the acrylamide derivative are screened, and the signal intensity curves of the ion pairs formed by the precursor ion and the characteristic daughter ions are recorded as a function of time. These curves constitute the mass spectrometry response data.

[0032] In the specific implementation, the process of obtaining the gas chromatographic elution signal and generating mass spectrometry response data is illustrated using the derivatized sample obtained in the previous embodiment. A gas chromatography-tandem mass spectrometry (GC-MS) system equipped with an autosampler is used. The sample vial containing the derivatized sample is placed in the sample tray of the autosampler. The autosampler program is set to splitless injection mode with an injection volume of 1.0 μL. The derivatized sample is injected into the chromatographic column through the inlet of the gas chromatography system. The inlet temperature is set to 250°C, the carrier gas is high-purity helium, and the constant flow rate mode is set to 1.0 mL / min. The preset gas chromatography temperature program is executed. The preset gas chromatography temperature program sets the initial column oven temperature to 50°C, holds for 1 minute, increases to 150°C at a rate of 20°C / min, then increases to 180°C at a rate of 5°C / min, and finally holds for 3 minutes. During the operation of the preset gas chromatography temperature program, the components in the derivatized sample migrate at different rates in the chromatographic column due to differences in volatility, and are eventually separated and elute sequentially. At the end of the chromatographic column, the elution signal changes over time by a detector. The elution signal is the gas chromatography elution signal.

[0033] In some embodiments, the gas chromatography temperature program can be adjusted, for example, by using different initial temperatures, heating rates, or final temperatures, to achieve optimal separation of the target analyte from interfering substances in a complex matrix. In some embodiments, to evaluate the effect of different temperature programs on the separation of acrylamide trifluoroacetate derivatives, different temperature gradients can be applied to the same derivatized sample while keeping other conditions constant, and the chromatographic retention time and peak shape of the target analyte can be recorded. The chromatographic separation effect can be assessed by resolution. The quantitative evaluation is based on the retention time and peak width of the target chromatographic peak and adjacent interfering peaks, and the relationship is as follows: in: Indicates the degree of separation; Indicates the retention time of the chromatographic peak of the target analyte, acrylamide derivative; Indicates the retention time of the main interfering chromatographic peak closest to the target peak; Indicates the peak width at the base of the chromatographic peak of the target analyte; This indicates the peak width at the base of the interfering chromatographic peak. See Table 2 for a comparison of separation performance under different temperature programs.

[0034] Table 2: Effect of different gas chromatography temperature programs on the resolution of acrylamide derivatives Optionally, different types of chromatographic columns can be used in the chromatographic separation process. For example, a capillary column with a 5% phenyl-95% dimethyl polysiloxane stationary phase, a length of 30 meters, an inner diameter of 0.25 millimeters, and a film thickness of 0.25 micrometers can be selected. It is understandable that the size of the chromatographic column and the properties of the stationary phase directly affect the retention and separation behavior of the components. Selecting a stationary phase with appropriate polarity helps to improve the separation of acrylamide derivatives from the co-eluting matrix.

[0035] In practice, the gas chromatography (GC) elution signal is imported into a tandem mass spectrometer (MS / MS), and ion pair signals corresponding to the characteristics of acrylamide-derived compounds are acquired in multiple reaction monitoring (MRM) mode. The GC elution is passed through a transfer line maintained at 280°C, and the GC elution signal is directly imported into the ion source of the MS / MS via an interface. The ion source performs electron bombardment ionization on the elution at an ionization energy of 70 eV, with the ion source temperature set to 230°C. In the first-stage mass analyzer of the MS / MS, the precursor ion corresponding to the trifluoroacetate derivative of acrylamide is selected, with a mass-to-charge ratio (m / z) of 127. The selected precursor ion is introduced into a collision cell and undergoes fragmentation upon collision with high-purity argon gas, with the collision energy optimized to 10 eV, generating daughter ions. In the second-stage mass analyzer of the MS / MS, pre-defined characteristic daughter ions related to the structural features of acrylamide-derived compounds are screened, for example, daughter ions with mass-to-charge ratios of 55 and 84 are selected for monitoring. The signal intensity curves of the ion pairs consisting of the precursor ion and the characteristic daughter ion are recorded as a function of time, and these curves constitute the mass spectrometry response data. It can be understood that multiple reaction monitoring (MRM) mode, by selectively monitoring the precursor ion-daughter ion pairs, can significantly reduce background chemical noise and improve the selectivity and sensitivity of the detection. Optionally, for acrylamide derivatives labeled with carbon-13 isotope as internal standards, the mass-to-charge ratio of the precursor ion is set to 130, and its characteristic daughter ion is monitored, with signals acquired synchronously under the same mass spectrometry conditions.

[0036] See Figure 4 This is a line graph of the gas chromatography column oven temperature program, fully illustrating the column temperature program in an acrylamide detection experiment. It clearly shows the three-stage temperature program: initial isothermal—programmed temperature—final temperature hold, completely replicating the chromatographic separation conditions for acrylamide derivatives. The initial low temperature (50℃) is used to retain weakly volatile components, preventing premature elution and peak overlap. Linear temperature increase (approximately 18.57℃ / min) achieves gradient separation of different volatile derivatives, improving chromatographic peak resolution. The final high temperature (180℃) is used for rapid elution of high-boiling-point residues, cleaning the column and preparing for the next injection. When chromatographic peak shapes are abnormal or retention times drift, comparing this graph can quickly pinpoint whether the temperature program setting is incorrect. It provides a baseline comparison for subsequent adjustments to parameters such as heating rate and isothermal time, intuitively assessing the impact of different programs on separation efficiency and detection time.

[0037] In one embodiment of the present invention, based on the known concentration of the stable isotope internal standard added to the internally standardized sample extract, the mass spectrometry response data is calibrated using an internal standard to calculate the relative response intensity of the target analyte. The signal peak area of ​​the characteristic daughter ion corresponding to the acrylamide-derived compound is extracted from the mass spectrometry response data and recorded as the target analyte peak area. The signal peak area of ​​the characteristic daughter ion corresponding to the carbon-13 isotope-labeled acrylamide-derived compound is extracted from the mass spectrometry response data and recorded as the target internal standard peak area. The ratio of the target analyte peak area to the target internal standard peak area is calculated to obtain the relative response intensity, which is used for subsequent calculations. Based on a pre-established standard curve, the relative response intensity is converted into a numerical value of acrylamide content in the fried food sample to be tested. The pre-established standard curve is obtained; the standard curve is a calibration curve plotted with the relative response intensity of acrylamide standard solutions of different concentrations as the ordinate and their corresponding concentrations as the abscissa. The relative response intensity of the acrylamide-derived compound in the fried food sample to be tested is substituted into the mathematical relationship of the standard curve, and the mathematical relationship is solved to calculate the concentration of acrylamide in the internal standard sample extract. Combining the sampling amount of the fried food sample to be tested and the final volume of the sample extract, the calculated concentration of acrylamide in the internal standard sample extract is converted into the content of acrylamide in the fried food sample to be tested. The pre-established standard curve is established by: preparing a series of acrylamide standard solutions with different known concentrations, adding a fixed amount of the stable isotope internal standard to each acrylamide standard solution to form a series of internal standard solutions. For each internal standard solution in the series of internal standard solutions, the same derivatization treatment, gas chromatography separation, and tandem mass spectrometry detection procedure as for the internal standard sample extract is performed. The mass spectrometry response of each internal standard solution in the series of internal standard solutions is recorded, and the relative response intensity of each internal standard solution is calculated. Using the concentration of the acrylamide standard solution as the abscissa and its corresponding relative response intensity as the ordinate, a linear regression fitting was performed to obtain the standard curve and its mathematical relationship.

[0038] In practical implementation, the process of internal standard calibration and calculation of the relative response intensity of the target analyte for mass spectrometry response data is illustrated using multiple reaction monitoring (MRC) chromatogram data of a derivatized sample from a potato chip sample as an example. In the mass spectrometry data processing software, the extracted ion chromatogram of the characteristic ion (mass-to-charge ratio 55) corresponding to the acrylamide derivatized compound is retrieved. The software automatically identifies and integrates the target chromatographic peak, extracting the signal peak area of ​​the characteristic ion corresponding to the acrylamide derivatized compound from the mass spectrometry response data, which is recorded as the target peak area. Assuming the integrated target peak area value is 12500, the same data processing interface is used. The extracted ion chromatogram of the characteristic ion (mass-to-charge ratio 58) corresponding to the carbon-13 isotope-labeled acrylamide derivatized compound is retrieved. The software automatically identifies and integrates the internal standard chromatographic peak, extracting the signal peak area of ​​the characteristic ion corresponding to the carbon-13 isotope-labeled acrylamide derivatized compound from the mass spectrometry response data, which is recorded as the target internal standard peak area. Assuming the integrated target internal standard peak area value is 11800, the ratio of the target peak area to the target internal standard peak area is calculated to obtain the relative response intensity. The calculation relationship is as follows: in: Represents the relative response intensity, and is a dimensionless ratio; This represents the signal peak area of ​​the characteristic daughter ions of acrylamide-derived compounds extracted from mass spectrometry response data; This represents the signal peak area of ​​the characteristic daughter ion of the carbon-13 isotope-labeled acrylamide derivative extracted from the mass spectrometry response data. Substitute this into the example numerical calculation. It is understandable that this calculation process is performed in every sample analysis, and the relative response intensity... This eliminates the impact of minor fluctuations in injection volume, short-term instrument response drift, or batch-to-batch variations in derivatization efficiency on absolute peak area. In some embodiments, when extracting the target analyte peak area and the target internal standard peak area from mass spectrometry response data, a manual integration method can be used, where the operator manually sets the start and end points and baseline of the chromatographic peaks to address complex baseline scenarios where automatic integration is inaccurate. In some embodiments, if the target analyte or internal standard has multiple characteristic daughter ion channels, the peak area of ​​the daughter ion channel with the highest signal-to-noise ratio can be selected for calculation, or the average peak areas of multiple daughter ion channels can be used for substituting into the calculation.

[0039] Based on a pre-established standard curve, the relative response intensity is converted into a numerical value of acrylamide content in the fried food sample to be tested. A pre-established standard curve is obtained, which is a calibration curve plotted with the relative response intensity of acrylamide standard solutions of different concentrations as the ordinate and their corresponding concentrations as the abscissa. The relative response intensity corresponding to the acrylamide derivative compounds in the fried food sample to be tested is substituted into the mathematical relationship of the standard curve. Assuming the mathematical relationship of the standard curve established for acrylamide trifluoroacetate derivatives is: in: Represents relative response intensity , The concentration of the acrylamide standard solution is represented in nanograms per milliliter. The calculated relative response intensity of the sample... Substitute the relation Solve the mathematical relationship to calculate the concentration of acrylamide in the extract of the internal standard sample. Nanograms per milliliter. Combining the sample weight of the fried food sample to be tested and the final volume of the sample extract, the calculated concentration of acrylamide in the internal standard sample extract is converted into the acrylamide content of the fried food sample to be tested. Assuming the potato chip sample used to prepare the extract is 5.00 grams and the final volume of the sample extract after pretreatment is 1.00 milliliters, then the acrylamide content in the sample is... It is understandable that this conversion process is based on the law of conservation of mass and the definition of concentration, and traces the concentration of the solution measured by the instrument back to the content of the original solid sample.

[0040] Optionally, a pre-established standard curve can be constructed by preparing a series of acrylamide standard solutions with different known concentrations. For example, acrylamide standards are accurately weighed and serially diluted with solvent to prepare acrylamide standard solutions with concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 nanograms per milliliter. A fixed amount of stable isotope internal standard is added to each acrylamide standard solution. For instance, 20.0 μL of a carbon-13 isotope-labeled acrylamide stock solution with a concentration of 1.0 μg per milliliter is added to each milliliter of standard solution, forming a series of internal standard solutions. For each internal standard solution in the series, the same derivatization, gas chromatography separation, and tandem mass spectrometry detection procedures as for the internal standard sample extract are performed. The mass spectrometric response of each internal standard solution in the series is recorded, and the relative response intensity of each internal standard solution is calculated based on the aforementioned peak area extraction and ratio calculation methods. Plotting the concentration of the acrylamide standard solution on the x-axis and its corresponding relative response intensity on the y-axis, the data points (0.1, 0.110), (0.5, 0.505), (1.0, 0.997), (2.0, 1.982), and (5.0, 4.940) were input into data processing software for linear regression fitting to obtain the standard curve and its mathematical relationship. linear correlation coefficient The value is 0.9998. Optionally, the linear regression fit can be calculated using the least squares method.

[0041] See Figure 5 This is a peak area comparison curve of the target analyte and the internal standard. The retention times of the target analyte and the internal standard highly overlap, proving that the isotopic internal standard behaves consistent with the target analyte, ensuring quantitative accuracy. The sharp peak shape and stable baseline indicate effective separation of the derivatized product from matrix interferences, demonstrating good method specificity. The peak signal intensity exceeds 12000, indicating a high signal-to-noise ratio, proving that the detection method has sufficient sensitivity to meet the needs of low-content acrylamide detection. It can serve as a control benchmark for parallel experiments to evaluate the stability of detection results between different batches of samples and different operators. When quantitative results are abnormal, the problem can be quickly located by comparing peak shape, retention time, and peak area. The sharp and symmetrical peak shape and stable, interference-free baseline indicate effective separation of the derivatized product from matrix impurities, demonstrating good method specificity and accurate identification of the target analyte.

[0042] The above embodiments are only used to illustrate the technical methods of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical methods of the present invention without departing from the spirit and scope of the technical methods of the present invention.

Claims

1. A method for detecting acrylamide content in fried foods, characterized in that, The method includes: A homogenization solution was prepared from the fried food sample to form the extract to be tested; Liquid-liquid extraction was performed on the extract to be tested to separate the extract phase containing the target analyte, resulting in a sample extract. A stable isotope internal standard is added to the sample extract to form an internally standardized sample extract. The extract of the internal standard sample is subjected to derivatization treatment to convert the target analyte into a derivatized compound with specific volatility, thereby generating a derivatized sample; The derivatized sample is injected into a gas chromatography system, and the components are separated based on a preset gas chromatography temperature program to obtain a gas chromatography elution signal. The gas chromatographic elution signal is imported into a tandem mass spectrometer, and ion pair signals corresponding to the characteristics of acrylamide-derived compounds are collected in multiple reaction monitoring mode to form mass spectrometry response data. Based on the known concentration of the stable isotope internal standard added to the internal standard sample extract, the mass spectrometry response data is calibrated using the internal standard, and the relative response intensity of the target analyte is calculated. Based on a pre-established standard curve, the relative response intensity is converted into the acrylamide content value in the fried food sample to be tested.

2. The method for detecting acrylamide content in fried foods according to claim 1, characterized in that, Liquid-liquid extraction is performed on the extract to be tested to separate the extract phase containing the target analyte, resulting in a sample extract comprising: The extract to be tested was transferred to a separatory container and a non-polar organic extraction solvent was added. The extract to be tested containing a non-polar organic extraction solvent was shaken and mixed, and then allowed to stand to separate into layers, forming a liquid-liquid system consisting of an upper aqueous phase and a lower organic phase. Collect the lower organic phase, which is an extract phase containing acrylamide; The collected extract phase was concentrated by rotary evaporation to remove some of the organic solvent, resulting in a concentrated sample extract.

3. The method for detecting acrylamide content in fried foods according to claim 2, characterized in that, Prior to the rotary evaporation concentration of the collected extract phase, the method further includes: The lower organic phase is passed through a chromatography column packed with a desiccant to remove residual moisture, resulting in a dry extract phase. The rotary evaporation concentration process is performed on the dried extract phase.

4. The method for detecting acrylamide content in fried foods according to claim 3, characterized in that, A stable isotope internal standard is added to the sample extract to form an internally standardized sample extract, comprising: The stable isotope internal standard is an acrylamide solution labeled with carbon-13 isotope; Accurately transfer a fixed volume of carbon-13 isotope-labeled acrylamide stock solution using a micropipette; The carbon-13 isotope-labeled acrylamide stock solution was injected into the sample extract to obtain a mixed solution; The mixed solution is vortexed to ensure that the carbon-13 isotope-labeled acrylamide is evenly distributed in the mixed solution, thereby forming the internal standard sample extract.

5. The method for detecting acrylamide content in fried foods according to claim 4, characterized in that, The extract of the internal standard sample is derivatized to convert the target analyte into a derivatized compound with specific volatility, generating a derivatized sample, including: Add trifluoroacetic anhydride derivatization reagent to the extract of the internally standardized sample; The extract of the internal standard sample after adding trifluoroacetic anhydride derivatization reagent was heated in a constant temperature water bath to promote the reaction of acrylamide with the trifluoroacetic anhydride derivatization reagent to generate a trifluoroacetic acid derivative of acrylamide. The reaction mixture after water bath heating was subjected to nitrogen blowing to remove excess derivatization reagents and byproducts, resulting in the derivatized sample with the trifluoroacetate derivative of acrylamide as the main analytical target.

6. The method for detecting acrylamide content in fried foods according to claim 5, characterized in that, The derivatized sample is injected into a gas chromatography system, and component separation is performed based on a preset gas chromatography temperature program to obtain a gas chromatography elution signal, including: The derivatized sample was injected into the column from the injection port of the gas chromatography system using a splitless injection mode. The preset gas chromatography temperature program is executed, which includes the setting of initial column oven temperature, programmed temperature ramp rate and final column oven temperature. During the operation of the preset gas chromatography temperature program, the components in the derivatized sample migrate at different rates in the chromatographic column due to differences in volatility, and are eventually separated and eluted sequentially. At the end of the chromatographic column, a detector records the effluent signal that changes over time; the effluent signal is the gas chromatographic effluent signal.

7. The method for detecting acrylamide content in fried foods according to claim 6, characterized in that, The gas chromatographic elution signal is imported into a tandem mass spectrometer, and ion pair signals corresponding to the characteristics of acrylamide-derived compounds are acquired in multiple reaction monitoring mode to form mass spectrometry response data, including: The gas chromatographic elution signal is directly imported into the ion source of the tandem mass spectrometer via an interface, and the ion source performs electron bombardment ionization on the elution. In the first-stage mass analyzer of the tandem mass spectrometer, the parent ion corresponding to the trifluoroacetate derivative of the acrylamide is selected; The selected parent ion is introduced into the collision cell and undergoes cleavage upon collision with the inert gas to produce daughter ions; In the second-stage mass analyzer of the tandem mass spectrometer, pre-defined characteristic ions related to the structural features of acrylamide-derived compounds are screened. The curves of the signal intensity of the ion pair composed of the parent ion and the characteristic daughter ion changing over time are recorded, and the curves constitute the mass spectrometry response data.

8. The method for detecting acrylamide content in fried foods according to claim 7, characterized in that, Based on the known concentration of the stable isotope internal standard added to the internally standardized sample extract, the mass spectrometry response data is calibrated using the internal standard, and the relative response intensity of the target analyte is calculated, including: From the mass spectrometry response data, the signal peak area of ​​the characteristic daughter ion corresponding to the acrylamide derivative compound is extracted and recorded as the target peak area; From the mass spectrometry response data, the signal peak area of ​​the characteristic daughter ion corresponding to the carbon-13 isotope-labeled acrylamide derivative compound is extracted and recorded as the target internal standard peak area; The relative response intensity is obtained by calculating the ratio of the peak area of ​​the target substance to the peak area of ​​the target internal standard. The relative response intensity eliminates the influence of injection volume fluctuation and instrument response drift.

9. The method for detecting acrylamide content in fried foods according to claim 8, characterized in that, Based on a pre-established standard curve, the relative response intensity is converted into a numerical value of acrylamide content in the fried food sample to be tested, including: Obtain the pre-established standard curve, which is a calibration curve plotted with the relative response intensity of acrylamide standard solutions of different concentrations as the ordinate and their corresponding concentrations as the abscissa. Substitute the relative response intensity corresponding to the acrylamide derivative compound in the fried food sample to be tested into the mathematical relationship of the standard curve; Solve the mathematical relationship to calculate the concentration of acrylamide in the extract of the internally standardized sample; By combining the sample size of the fried food sample to be tested and the final volume of the sample extract, the calculated concentration of acrylamide in the internal standard sample extract is converted into the content of acrylamide in the fried food sample to be tested.

10. The method for detecting acrylamide content in fried foods according to claim 9, characterized in that, The pre-established standard curve is established through a process that includes: Prepare a series of acrylamide standard solutions with different known concentrations; A fixed amount of the stable isotope internal standard is added to each of the acrylamide standard solutions to form a series of internally standardized standard solutions. For each internal standard solution in the series of internal standard solutions, the same derivatization, gas chromatography separation and tandem mass spectrometry detection process as that of the internal standard sample extract is performed. Record the mass spectrometry response of each internal standard solution in the series of internal standard solutions, and calculate the relative response intensity of each internal standard solution; Using the concentration of the acrylamide standard solution as the abscissa and its corresponding relative response intensity as the ordinate, a linear regression fitting was performed to obtain the standard curve and its mathematical relationship.

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