A method for simultaneously detecting 17 types of heat-processing hazards in 4 categories and its application

Abstract

This invention provides a method for simultaneously detecting 17 heat-processing hazards in 4 categories, comprising: adding n-hexane to the sample, mixing thoroughly, discarding the n-hexane phase, extracting acrylamide, 5-hydroxymethylfurfural, and free heterocyclic amines using dimethyl ketone and ultrasound-assisted extraction, collecting the supernatant, and purifying it; simultaneously transferring the precipitate to another tube, reducing it with sodium tetrahydroborate, hydrolyzing it with hydrochloric acid to release bound heterocyclic amines and late-stage glycosylation products, and performing UPLC-MS / MS analysis. This invention is the first to propose a two-step extraction method for simultaneously determining 17 heat-processing hazards in 4 categories within 7 minutes using UPLC-MS / MS. The accuracy, precision, detection limit, quantitation limit, and recovery rate of the method were verified, and all test indicators met the requirements. This method is applicable to the determination of heat-processing hazards in four types of food matrices: high starch, high protein, high fat, and high sugar.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for simultaneously detecting 17 kinds of heat-processing hazards in 4 categories in heat-processed foods. Background Technology

[0002] During food heat processing, the Maillard reaction imparts color and flavor to food. However, it also easily generates harmful byproducts of heat processing that threaten human health, such as acrylamide (AA), 5-hydroxymethylfurfural (HMF), heterocyclic amines (HAs), and advanced glycation end products (AGEs). These are potentially harmful Maillard reaction products in food and herb medicines. (Li et al.) Journal of Food Quality , 2021 Acrylamide (AA) is commonly found in carbohydrate-rich foods and has also been detected in high-protein and high-sugar foods. AA possesses neurotoxicity and genotoxicity and has been classified as a probable human carcinogen (Group 2A) by the International Agency for Research on Cancer (IARC) (Acrylamide from Maillard reaction products. Stadler et al., Nature , 419(6906), 449–450.). HMF is found in high concentrations in dried fruit, caramel, and bread, and its metabolites in the body are mutagenic and carcinogenic (Evolution of 5-hydroxymethylfurfural (HMF) and furfural (F) in fortified wines submitted to overheating conditions. Pereira et al., Food Research International, 44(1), 71–76.). HAs are typically produced in high-protein foods heated (above 150°C). To date, 25 different HAs have been identified in food. Of these, 9 are classified as possible human carcinogens (Group 2B) by the IARC, and 1 is classified as Group 2A. High levels of AGEs are found in baked and fried foods. Long-term consumption of foods high in AGEs can promote oxidative stress and inflammation, inducing various chronic diseases such as diabetes, metabolic disorders, and cancer. These heat-processed byproducts that threaten human health often coexist in food. Therefore, a detection method is needed to simultaneously detect heat-processing-related hazards such as AA, HMF, HAs, and AGEs in complex food matrices in order to monitor potential safety risks in food.

[0003] On the one hand, HAs and AGEs exist in both free and protein-bound states, but the protein-bound state cannot be directly detected and must first be converted to the free state. During this conversion, the differences between HAs and AGEs must be considered. Furthermore, these substances exhibit significant polarity differences, making simultaneous extraction difficult. In addition, food matrices rich in lipids and proteins can interfere with the extraction and detection of target analytes, further increasing the difficulty of simultaneous extraction and detection. To date, there are no reports on the simultaneous detection of AA, HMF, HAs, and AGEs. Summary of the Invention

[0004] To address the problem of multiple hazardous substances coexisting in heat-processed foods, this invention proposes a detection method based on a two-step extraction method and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to simultaneously detect 17 kinds of heat-processing hazardous substances in 4 categories, specifically acrylamide, 5-hydroxymethylfurfural, 13 heterocyclic amines, and 2 late glycosylation terminates.

[0005] The technical solution for achieving the above-mentioned objective of this invention is as follows:

[0006] A method for simultaneously detecting 17 types of heat-processing hazards in 4 categories, wherein the 17 types of heat-processing hazards in 4 categories include: acrylamide, 5-hydroxymethylfurfural, and 13 heterocyclic amines: 2-amino-3-methylimidazolium[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazolium[4,5-f]quinoline (MeIQx), 1-methyl-9H-pyridine[3,4-b]indole (Harman), 9H-pyridine[3,4-b]indole (Norharman), 2-amino-9H-pyridine[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyridine[2,3-b]indole (MeAαC), and 3-amino-1,4-dimethyl-5H-pyridine[4,3-b]indoleacetic acid. The method comprises the following steps: ester (Trp-P-1), 3-amino-1-methyl-5H-pyridinium[4,3-b]indoleacetic acid ester (Trp-P-2), 2-amino-6-methyldipyridano[1,2-α:3'2'-d]imidazolium hydrochloride (Glu-P-1), 2-aminodipyridano[1,2-α:3'2'-d]imidazolium hydrochloride (Glu-P-2), 2-amino-1-methyl-6-benzimidazol[4,5-b]pyridine (PhIP), 2-amino-1,6-dimethylimidazol[4,5-b]pyridine (DMIP), 2-amino-5-phenylpyridine (Phe-P-1), and two late glycosylation terminal products: carboxymethyl lysine (CML) and carboxyethyl lysine (CEL).

[0007] S1: Add n-hexane to the sample, vortex to mix, sonicate, centrifuge, and discard the n-hexane phase to remove lipids; add dimethyl ketone, use ultrasound to extract acrylamide, 5-hydroxymethylfurfural and free heterocyclic amines, centrifuge, collect the supernatant of the dimethyl ketone layer, add the mixed internal standard solution, purify and dry, and redissolve with the initial mobile phase;

[0008] S2: Transfer the precipitate remaining after centrifugation in step S1 to another tube, add sodium tetrahydroborate for reduction; add hydrochloric acid for hydrolysis; add mixed internal standard solution to the hydrolysis product, pass through a solid phase extraction column (Oasis MCX), elute with ammoniated methanol, dry, and reconstitute with the initial mobile phase;

[0009] S3: Prepare a series of mixed standard working solutions of different concentrations and establish standard curves for 17 substances; analyze the products obtained by reconstitution in steps S1 and S2 separately or in combination using UPLC-MS / MS and quantify using the internal standard method.

[0010] The product from the reconstitution in step S1 is used to detect acrylamide, 5-hydroxymethylfurfural, 13 heterocyclic amines (AA, HMF and 13 free HAs); the product from the reconstitution in step S2 is used to detect 13 bound HAs and 2 bound AGEs.

[0011] The following are preferred technical solutions of the present invention.

[0012] In step S1, the sample is a heat-processed food sample to be tested. 10 mL of n-hexane is added per gram of sample, the sample is vortexed, sonicated for 5 to 15 minutes, and then centrifuged to discard the n-hexane phase in order to remove lipids.

[0013] This step can be repeated three times.

[0014] In step S1, 10 mL of dimethyl ketone is added to each gram of sample and vortexed for 1–2 minutes. After ultrasonic extraction for 10–30 minutes, the mixture is continuously centrifuged at 5000–20000 rpm for 5–20 minutes.

[0015] In step S1, the final concentration of the mixed internal standard solution is 200-1000 μg / L; the added purification substances are anhydrous magnesium sulfate (MgSO4) and N-propylethylenediamine (primary secondary amine, PSA).

[0016] Optionally, the volume of the mixed internal standard solution added is 20 μL per gram of sample, and the concentration of the internal standard solution is 50 mg / L. 13 C3-AA, 10 mg / L 13 C6-HMF and 10 mg / L 4,7,8-TriMeIQx. After adding the mixed internal standard solution, the solution was purified with anhydrous MgSO4 and PSA.

[0017] Add internal standards corresponding to AA, HMF, and HAs. 13 C3-AA 13 C6-HMF and 4,7,8-TriMeIQx serve two purposes: firstly, to eliminate the interference of matrix effects, reduce masking or enhancement effects that could lead to underestimation or overestimation of content; and secondly, to correct for losses caused by the pretreatment process, thus avoiding underestimation of content.

[0018] The role of anhydrous MgSO4 is to adsorb moisture in the sample, promote the transfer of target analytes to the organic phase, and improve extraction efficiency; the role of PSA is to adsorb some impurities and purify the extract. The amount of each of the two added per gram of sample can be 4.0 g and 0.03 g, respectively.

[0019] In step S2, the precipitate remaining after centrifugation is transferred to a polytetrafluoroethylene tube for analysis of bound HAs and AGEs. This method can simultaneously convert bound HAs and AGEs, achieving accurate quantification and reducing the time cost of pretreatment and the input of organic reagents.

[0020] In step S2, the reduction process involves treating the solution with a pH 9.2 borate buffer and 1M sodium tetrahydroborate (dissolved in 0.1M sodium hydroxide) for 10–15 hours. The reduced solution is then used to precipitate the protein using a chloroform-methanol mixture.

[0021] This reduction step aims to reduce fructose lysine, thereby preventing the formation of new AGEs during acid hydrolysis and resulting in an overestimation of its content.

[0022] The specific procedure can be as follows: Weigh 1g of sample and reduce it at 4°C for 12 hours using 10mL of borate buffer (0.2M, pH 9.2) and 5mL of sodium tetrahydroborate (1M, dissolved in 0.1M sodium hydroxide solution). The reduction solution is preferably a 10mL chloroform-methanol (2:1, v / v) mixture to precipitate the protein.

[0023] In step S2, the hydrolysis process is as follows: hydrolyze in 6M hydrochloric acid for 20–28 hours. After cooling to room temperature, filter the protein hydrolysate and dilute with ultrapure water.

[0024] More preferably, the hydrolysis conditions are: adding 10 mL of 6M hydrochloric acid and hydrolyzing at 110°C for 24 hours.

[0025] The solid-phase extraction column can be an Oasis MCX or an existing adsorption device in the art capable of extracting compounds with cation exchange groups. The concentration of ammoniated methanol used for elution is 1–10% (v / v).

[0026] In step S2, the final concentration of the mixed internal standard solution is 200–400 μg / L.

[0027] More preferably, the volume of the mixed internal standard solution added is 20 μL per gram of sample, and the concentrations of the internal standard solutions are 10 mg / L 4,7,8-TriMeIQx, 20 mg / L D4-CML, and 20 mg / L D4-CEL, respectively.

[0028] The purpose of adding internal standard products here is the same as above.

[0029] In steps S1 and S2, the initial mobile phase used for resolution is 1% methanol-formic acid water, v / v.

[0030] In existing technologies, water / acetonitrile (1:1, v / v) is generally used for dissolution after nitrogen blowing, while this method uses the initial mobile phase for redissolution, which can reduce the influence of solvent effect during sample injection compared with conventional methods.

[0031] In step S3, the preferred UPLC conditions before UPLC-MS / MS analysis are:

[0032] Column: BEH C18 column; Mobile phase: 0.1% formic acid water (v / v) (A) and methanol (B); Gradient elution: 99% A, 0–0.5 min; 99–75% A, 0.5–1.5 min; 75–62% A, 1.5–2.5 min; 62–55% A, 2.5–3.5 min; 55–52% A, 3.5–4.0 min; 52–46% A, 4.0–5.0 min; 46–35% A, 5.0–5.9 min; 35–99% A, 5.9–7.0 min.

[0033] Optionally, the UPLC run time is 7 minutes; flow rate: 0.3 mL / min; column temperature: 30 °C; injection volume: 1 μL.

[0034] The preferred MS / MS conditions are:

[0035] ESI: Positive ion mode; Capillary voltage: 0.5kV; Ion source temperature: 100℃; Desolventizing temperature: 500℃; Conical gas flow rate: 150L / h; Desolventizing gas flow rate: 1000L / h; Collision gas flow rate: 0.12mL / min.

[0036] The UPLC-MS / MS analysis can be performed using Acquity UPLC. TM The I-Class system and Xevo TQ-S triple quadrupole mass spectrometer can be used in combination, or other existing ultra-high performance liquid chromatography-mass spectrometry systems in the field.

[0037] The beneficial effects of this invention are as follows:

[0038] 1. This invention proposes a two-step extraction method that is simple and efficient, capable of extracting 17 kinds of heat-related hazardous substances from 4 categories from actual samples in a single operation. Conventional methods can only extract single or small amounts of hazardous substances. Compared with existing methods, this method extracts a wider variety of hazardous substances and reduces pretreatment time by more than 50%.

[0039] 2. Pre-processing can easily lead to the secondary generation or loss of hazardous substances, resulting in overestimation or underestimation of their content. In this invention, corresponding corrective measures are adopted to ensure the accurate quantification of heat-processed hazardous substances in food.

[0040] 3. This invention enables simultaneous detection within 7 minutes using UPLC-MS / MS. Using a BEH C18 column and a mobile phase of 0.1% formic acid-water and methanol, 17 analytes and 5 internal standards can be completely separated. The method's accuracy is 98.13–100.96%, the detection limit is 0.01–0.89 μg / L, and the quantitation limit is 0.02–2.96 μg / L. Almost all analytes exhibit high correlation coefficients (R²) within their respective linear ranges. 2>0.999). Intra-day precision and inter-day precision were 0.25–9.90% and 0.17–12.43%, respectively.

[0041] 4. This method can be applied to a variety of food matrices, including high-starch, high-protein, high-fat, and high-sugar foods. Attached Figure Description

[0042] Figure 1 Chromatograms of 17 target analytes and 5 internal standards under optimized conditions: 1: CML and D4-CML; 2: CEL and D4-CEL; 3: AA and 13 C3-AA; 4: IQ; 5: DMIP; 6: Glu-P-2; 7: HMF and 13 C6-HMF; 8: Glu-P-1; 9: MeIQx; 10: Norharman; 11: Phe-P-1; 12: Harman; 13: 4,7,8-TriMeIQx; 14: PhIP; 15: Trp-P-2; 16: AαC; 17: Trp-P-1; 18: MeAαC.

[0043] Figure 2 Separation chromatogram of Acquity UPLC HSS T3 column.

[0044] Figure 3 Chromatogram of separation of 3.03 mM ammonium acetate.

[0045] Figure 4 Chromatograms showing the separation of methanol and acetonitrile.

[0046] Figure 5 MRM chromatograms of 17 target analytes and 5 internal standards.

[0047] Figure 6 : Flowchart of this detection method. Detailed Implementation

[0048] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0049] Unless otherwise specified, all technical means used in this instruction manual are known in the art. All raw materials used are commercially available.

[0050] The preparatory stage of this assay involves preparing the standard solutions:

[0051] S1: Dissolve AA, HMF and 13 HA standards in methanol; dissolve two AGE standards in ultrapure water to obtain standard stock solutions of 1 mg / mL.

[0052] A series of mixed standard working solutions containing AA, HMF, HAs and AGEs were prepared by stepwise dilution of the standard stock solution with the initial mobile phase for later use.

[0053] Example 1: Optimization of Mass Spectrometry (MS / MS) Conditions

[0054] To maximize the sensitivity and selectivity of the target analytes, standards were directly injected into the mass spectrometer to monitor and optimize MS / MS parameters. Based on the structural characteristics of the 17 analytes, ESI positive ion mode and multiple reaction monitoring (MRM) mode were selected. The responsiveness of molecular ions was improved by optimizing the capillary voltage and cone voltage. The collision voltage was optimized, and in the second mass spectrometry scan, the fragment ion with the highest response was selected for quantification, and the fragment ion with the second highest response for qualitative analysis. The optimized capillary voltage was 0.5 kV; ion source temperature was 100 °C; desolvation temperature was 500 °C; cone gas flow rate was 150 L / h; desolvation gas flow rate was 1000 L / h; and collision gas flow rate was 0.12 mL / min. The optimal MS / MS parameters for AA, HMF, AGEs, and HAs are shown in Table 1.

[0055] Table 1. Retention times and MS / MS parameters of 17 analytes and 5 internal standards

[0056]

[0057] a: Internal standard products

[0058] Example 2: Optimization of Up-Phase Liquid Chromatography (UPLC) Conditions:

[0059] Optimization of UPLC conditions is crucial for baseline separation and accurate quantification of 17 analytes.

[0060] Column optimization:

[0061] For these target analytes, commonly used chromatographic columns are the Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) and the Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). Therefore, this example tests and compares the separation performance of these two commonly used columns for the target analytes. The results show that on the HSS T3 column, some HAs exhibited poor peak shapes, such as MeAαC, Phe-P-1, Harman, and Norharman (…). Figure 2 On the BEH C18 column, the peaks of the 17 analytes were symmetrical and sharp. Therefore, the BEH C18 column was ultimately chosen.

[0062] Optimization of the mobile phase:

[0063] The choice of mobile phase helps improve the ionization and separation efficiency of analytes. Formic acid or ammonium salts are often added to the mobile phase to improve peak shape, sensitivity, and separation efficiency. Therefore, mobile phase B was fixed at acetonitrile, and the separation effects of two different mobile phases, 0.1% formic acid solution and 3.03 mM ammonium acetate (pH 4.0), were compared. When mobile phase A was 3.03 mM ammonium acetate, HAs such as IQ, MeIQx, Aαc, and MeAαC exhibited tailing peaks. Figure 3 However, when 0.1% formic acid solution was used as mobile phase A, the peak shape was sharp and symmetrical, with no tailing phenomenon. Therefore, 0.1% formic acid solution was ultimately used as mobile phase A.

[0064] Furthermore, with mobile phase A fixed at 0.1% formic acid-water, two different mobile phases, methanol and acetonitrile, were compared. The results showed that methanol provided better retention and separation efficiency for all analytes. Figure 4 Therefore, methanol is used as the mobile phase B.

[0065] In the optimized method, a BEH C18 column was selected, and the mobile phase consisted of 0.1% formic acid, water, and methanol. The gradient elution program was further optimized to improve the separation of 17 target analytes. To extend the retention times of highly polar analytes AA and AGEs, mobile phase A was started at 99%. The optimized gradient elution program was as follows: 99% A, 0–0.5 min; 99–75% A, 0.5–1.5 min; 75–62% A, 1.5–2.5 min; 62–55% A, 2.5–3.5 min; 55–52% A, 3.5–4.0 min; 52–46% A, 4.0–5.0 min; 46–35% A, 5.0–5.9 min; 35–99% A, 5.9–7.0 min. Finally, using a 7-minute gradient elution procedure, each target analyte and its internal standards were clearly eluted into a single peak without any interfering peaks, demonstrating the high specificity of the developed method. Figure 5 Chromatograms of 17 analytes under optimized conditions are shown below. Figure 1 As shown.

[0066] Example 3: Optimization of the pretreatment process

[0067] Optimization of extraction solvent:

[0068] The wide polarity range of these analytes makes finding suitable solvents for simultaneous extraction extremely challenging. In this example, AA, HMF, and 13 HA standards were added to a blank pork sample, and the extraction efficiency was evaluated by testing the recoveries after extraction with different solvents. First, the extraction efficiency of ultrapure water, acetonitrile, and methanol was tested. Acetonitrile and methanol are commonly used organic extraction solvents. Ultrapure water showed good extraction efficiency for AA and HMF, but its extraction efficiency was not ideal due to the moderate polarity of heterocyclic amines. Furthermore, ultrapure water easily extracts water-soluble substances from food, resulting in a viscous solution that is difficult to process in subsequent operations. Methanol extraction lacks specificity, easily extracting both target analytes and interfering substances simultaneously, which is detrimental to the reconstruction of analytes after nitrogen blowing. Acetonitrile showed good extraction efficiency for most analytes, but poor extraction efficiency for AA. After examining the solubility of AA in other organic solvents, the extraction efficiency of dimethyl ketone was tested, showing good recoveries for all target analytes. Dimethyl ketone was ultimately selected as the extraction solvent.

[0069] Optimization of acid hydrolysis:

[0070] Bound HAs and AGEs constitute a high proportion of food, but cannot be directly detected and must first be converted to their free state. Acid hydrolysis can convert bound HAs and AGEs into their free state by hydrolyzing proteins. Acid hydrolysis tests on raw pork samples without analytes revealed that the process did not produce new HAs, but it did generate CML and CEL, leading to an overestimation of AGEs content. Therefore, this method was improved by adding a reduction step before acid hydrolysis, adding 1M sodium tetrahydroborate to reduce Amadori products such as fructose-lysine and prevent the formation of new AGEs during acid hydrolysis. The reduction treatment effectively corrected the overestimation of AGEs content in the sample and confirmed that it did not interfere with the conversion of bound HAs. Subsequently, the acid stability of these analytes was examined, revealing that a small amount of HAs is acid-instable. The addition of corresponding internal standards corrected for losses during acid hydrolysis, avoiding underestimation of bound HAs and AGEs content. The optimized method can simultaneously convert bound HAs and AGEs without causing either overestimation or underestimation.

[0071] Example 4: Method Validation

[0072] The proposed method was validated according to the standards in SANTE / 11813 / 2017 regarding parameters such as linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, recovery, precision, matrix effect, and residue.

[0073] Linearity, LOD, LOQ, and accuracy verification:

[0074] The correlation coefficient (R) of the standard curve 2 Linearity was assessed using the calibration concentration as the independent variable (x) and the respective area ratio (compound area / respective internal standard area) as the dependent variable (y). LOD and LOQ were defined as the dilution concentrations with signal-to-noise ratios (S / N) of 3 and 10, respectively. Accuracy was assessed as the percentage difference between the observed concentration and the specified concentration. The range for AA is 31.25 to 2000 μg / L (31.25, 62.5, 125, 250, 500, 1000 and 2000 μg / L), the range for HMF is 3.9065 to 500 μg / L (3.9063, 7.8125, 15.625, 31.25, 625, 125 and 500 μg / L), and the ranges for CEL, CML, AαC, MeAαC, Norharman, Glu-P-1, Glu-P-2, Trp-P-1 and Trp-P-2 are 3.125 to 400 μg / L (3.0625, 6.125, 12.25, 25, 50, 100, 200 and 400 μg / L). The ranges for IQ, MeIQx, PhIP, DMIP, Phe-P-1, and Harman are 3.125 to 200 μg / L (3.0625, 6.125, 12.25, 25, 50, 100, and 200 μg / L). For each level, add 20 μL of the mixed internal standard solution (50 mg / L). 13 C3-AA, 10 mg / L 13 C6-HMF, 10 mg / L 4,7,8-TriMeIQx, 20 mg / L D4-CML, and 20 mg / L D4-CEL. Data were obtained using weighted least squares linear regression analysis with a weighting factor of 1 / x. All analytes showed good linearity within their respective ranges, with almost all coefficients of determination (R²) in the matrix. 2 All values ​​were greater than 0.999, LODs ranged from 0.01 to 0.89 μg / L, LOQs ranged from 0.02 to 2.96 μg / L, and the accuracy ranged from 98.13 to 100.96%.

[0075] Table 2: Methodological Evaluation of 17 Hazards

[0076]

[0077]

[0078] Validation of recovery rate, precision, matrix effect, and residue:

[0079] In four representative food categories—high-starch foods (biscuits), high-fat foods (roasted nuts), high-sugar foods (dried apricots), and high-protein foods (plant-based meat)—three concentration levels (50%, 100%, and 200%, m / m) of mixed standard working solutions were added to test the recovery rate of the method. Precision (intra-day and inter-day precision) was evaluated through repeated analysis of spiked samples at the three concentration levels (n=6, n=3) and expressed as relative standard deviation (RSD, %). Stable internal standards were used. 13 C3-AA 13 Matrix effects of analytes were corrected using C6-HMF, D4-CML, D4-CEL, and 4,7,8-TriMeIQx. Method residues were assessed by injecting two blanks after determining the highest standard concentration within the calibration range. Recovery rates for most analytes were in the range of 70–130%, with intra-day and inter-day precision of 0.25–9.90% and 0.17–12.43%, respectively, meeting the requirements. No analyte residues were observed in the two injected blanks.

[0080] In summary, this method has the advantages of high accuracy, low detection and quantitation limits, good linear correlation, good precision, and strong specificity.

[0081] Example 5: Application of the Method

[0082] This embodiment provides a method for simultaneously detecting 17 types of heat-processing hazards in 4 categories. The detection process is as follows: Figure 6 .

[0083] Four representative food matrices were selected: high-starch foods (biscuits and potato chips), high-protein foods (pork patties and plant-based meat), high-fat foods (roasted nuts), and high-sugar foods (dried apricots). The contents of AA, HMF, HAs, and AGEs were determined.

[0084] Weigh 1g of the pulverized food sample into 50mL centrifuge tubes and add 10mL of n-hexane. Vortex the tubes for 1 minute, sonicate for 10 minutes, and then centrifuge at 10,000 rpm for 10 minutes. Discard the hexane phase to remove lipids. Repeat this step three times. Then, add 10mL of dimethyl ketone to the centrifuge tubes and vortex for 1 minute. Use sonic-assisted extraction, and after 20 minutes, continuously centrifuge the mixture at 10,000 rpm for 10 minutes.

[0085] The supernatant of the dimethyl ketone layer was collected in another centrifuge tube for analysis of AA, HMF, and 13 free HAs. 20 μL of internal mixed standard solution (50 mg / L) was added. 13 C3-AA, 10 mg / L 13C6-HMF and 10 mg / L 4,7,8-TriMeIQx were then added. Anhydrous MgSO4 (4.0 g) and PSA (0.03 g) were added. The centrifuge tubes were sealed and vortexed immediately for 2 minutes. After centrifugation at 10,000 rpm for 10 minutes, the dimethyl ketone layer was transferred to a clean 10 mL centrifuge tube and dried under a gentle nitrogen stream. The residue was reconstituted with the initial mobile phase (1 mL, 1% methanol-formic acid water, v / v).

[0086] The precipitate was transferred to PTFE tubes for analysis of HAs and AGEs. The precipitate was reduced at 4°C for 12 hours using 10 mL borate buffer (0.2 M, pH 9.2) and 5 mL sodium tetrahydroborate (1 M, dissolved in 0.1 M sodium hydroxide solution). Then, 10 mL of chloroform-methanol (2:1, v / v) mixture was added to precipitate the protein. The mixture was centrifuged at 10,000 rpm for 10 min, followed by hydrolysis at 110°C for 24 hours using 10 mL of 6 M hydrochloric acid. After cooling to room temperature, the protein hydrolysate was filtered and diluted to 50 mL with ultrapure water. Next, 1 mL of the hydrolysate was taken and 20 μL of a mixed internal standard solution (10 mg / L 4,7,8-TriMeIQx, 20 mg / L D4-CML, and 20 mg / L D-CEL) was added. The mixed solution was loaded into a solid-phase extraction column (Oasis MCX, 3 mL / 60 mg, Waters) pre-activated with methanol. The column was then washed with water (3 mL) and methanol (3 mL), and finally eluted with 5% ammoniated methanol (6 mL, v / v). The eluent was dried under nitrogen and then reconstituted with the initial mobile phase (1 mL, 1% methanol-formic acid water, v / v).

[0087] Prior to UPLC-MS / MS analysis, all sample solutions were filtered through a 0.22 μm syringe filter. In this embodiment, the supernatant and the precipitate reconstitution solution were injected separately.

[0088] UPLC-MS / MS analysis conditions: The optimized mobile phase and gradient elution program of Example 2 were used, along with a BEH C18 column. The optimal MS / MS parameters listed in Example 1 were employed.

[0089] The test results are shown in Tables 3 and 4.

[0090] Table 3: Content of AA, HMF and Free Heterocyclic Amines in Food

[0091]

[0092] Note: nd, not detected; nq, not quantified.

[0093] Table 4: Content of bound heterocyclic amines and late-stage glycosylation end products in food

[0094]

[0095] Note: nd, not detected; nq, not quantified.

[0096] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A method for simultaneous detection of 17 heat processing hazards in 4 categories, characterized in that, The 17 types of heat-processing hazards in the four categories include: acrylamide, 5-hydroxymethylfurfural, and 13 heterocyclic amines: 2-amino-3-methylimidazolium[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazolium[4,5-f]quinoline (MeIQx), 1-methyl-9H-pyridine[3,4-b]indole (Harman), 9H-pyridine[3,4-b]indole (Norharman), 2-amino-9H-pyridine[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyridine[2,3-b]indole (MeAαC), 3-amino-1,4-dimethyl-5H-pyridine[4,3-b]indole acetate (Trp-P-1), 3-amino-1-methyl-5H-pyridine[ The following compounds were tested: [4,3-b]indole acetate (Trp-P-2), 2-amino-6-methyldipyridano[1,2-α:3'2'-d]imidazolium hydrochloride (Glu-P-1), 2-aminodipyridano[1,2-α:3'2'-d]imidazolium hydrochloride (Glu-P-2), 2-amino-1-methyl-6-benzylimidazole[4,5-b]pyridine (PhIP), 2-amino-1,6-dimethylimidazolium[4,5-b]pyridine (DMIP), 2-amino-5-phenylpyridine (Phe-P-1), and two late-stage glycosylation terminates: carboxymethyl lysine (CML) and carboxyethyl lysine (CEL). The samples tested included biscuits, potato chips, pork patties, plant-based meat, roasted nuts, and dried apricots. The method included the following steps: S1: Add n-hexane to the sample, vortex to mix, sonicate, centrifuge, and discard the n-hexane phase to remove lipids; add dimethyl ketone, and use ultrasound-assisted extraction to extract acrylamide, 5-hydroxymethylfurfural and free heterocyclic amines, centrifuge, collect the supernatant of the dimethyl ketone layer, add the mixed internal standard solution, add the purification substances anhydrous magnesium sulfate and N-propylethylenediamine, purify and dry, and redissolve with the initial mobile phase; S2: Transfer the precipitate remaining after centrifugation in step S1 to another tube, add sodium tetrahydroborate for reduction; add hydrochloric acid for hydrolysis; add a mixed internal standard solution to the hydrolysis product, pass through a solid-phase extraction column, elute with ammoniated methanol, dry, and reconstitute with the initial mobile phase; the mixed internal standard solution is D4-CEL, D4-CML, and 4,7,8-TriMeIQx, with a final concentration of 200~400 μg / L; the sodium tetrahydroborate reduction operation is as follows: treat with borate buffer at pH 9.2 and 1 M sodium tetrahydroborate solution for 10~15 hours, and precipitate the protein with a chloroform-methanol mixture; the solid-phase extraction column is Oasis MCX; In steps S1 and S2, the initial mobile phase used for reconstitution was 1% methanol-formic acid water, v / v ; S3: Prepare a series of mixed standard working solutions of different concentrations to establish standard curves for 17 substances; the products obtained by reconstitution in steps S1 and S2 are detected by ultra-high performance liquid chromatography-tandem mass spectrometry, and quantified by internal standard method. Column: BEH C18 column; Multiple reaction monitoring mode employed; The liquid phase conditions are: mobile phase A: 0.1% formic acid in water. v / v Mobile phase B: methanol; gradient elution: 99% A, 0–0.5 min; 99–75% A, 0.5–1.5 min; 75–62% A, 1.5–2.5 min; 62–55% A, 2.5–3.5 min; 55–52% A, 3.5–4.0 min; 52–46% A, 4.0–5.0 min; 46–35% A, 5.0–5.9 min; 35–99% A, 5.9–7.0 min; The mass spectrometry conditions were as follows: ESI: positive ion mode; capillary voltage: 0.5 kV; ion source temperature: 100 ℃; desolvation temperature: 500 ℃; cone gas flow rate: 150 L / h; desolvation gas flow rate: 1000 L / h; collision gas flow rate: 0.12 mL / min.

2. The method for simultaneously detecting 17 kinds of heat-processing hazards in 4 categories according to claim 1, characterized in that, In step S1, extract lipids by adding 10 mL of n-hexane per gram of sample; after vortexing, sonicate for 5-15 minutes, then centrifuge and discard the n-hexane phase to remove lipids.

3. The method for simultaneously detecting 17 kinds of heat-processing hazards in 4 categories according to claim 1, characterized in that, In step S1, 10 mL of dimethyl ketone is added to each gram of sample and vortexed for 1-2 minutes. After ultrasonic extraction for 10-30 minutes, the mixture is continuously centrifuged at 5000-20000 rpm for 5-20 minutes.

4. The method for simultaneously detecting 17 kinds of heat-processing hazards in 4 categories according to claim 1, characterized in that, In step S1, the mixed internal standard solution is 13 C3-AA, 13 C6-HMF and 4,7,8-TriMeIQx, final concentration 200-1000 μg / L.

5. The method for simultaneously detecting 17 kinds of heat-processing hazards in 4 categories according to claim 1, characterized in that, In step S2, the hydrolysis operation is as follows: hydrolyze in 6M hydrochloric acid for 20-28 hours.

Images