A method for enriching and detecting advanced glycation end products

Abstract

The application relates to an advanced glycation end product enrichment method and a detection method, and belongs to the technical field of chemical detection. The advanced glycation end product enrichment method of the application can realize selective and efficient enrichment of advanced glycation end products by adopting a magnetic solid-phase extraction agent to perform magnetic solid-phase extraction on the advanced glycation end products in a liquid sample containing the advanced glycation end products. The magnetic solid-phase extraction agent has high porosity, low crystal density, large specific surface area and good structural stability, can be quickly separated from the sample solution through an external magnet, has the advantages of simple operation, short extraction time, low solvent consumption and the like, and can overcome problems such as high cost of a solid-phase extraction column and complex operation process of passing through the column.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for enriching and detecting advanced glycation end products, and belongs to the technical field of chemical detection. BACKGROUND

[0002] Advanced glycosylation end products (AGEs) are chemical hazards formed by Maillard reaction of macromolecules such as proteins and amino acids under non-enzymatic conditions. Studies have shown that the accumulation of AGEs in the human body is closely related to the occurrence of many diseases, such as diabetic chronic complications, aging, atherosclerosis, hypertension, Alzheimer's disease, etc. Foods rich in carbohydrates, fats and proteins are prone to produce AGEs during thermal processing, such as baked foods, which are recognized as an important source of AGEs in diet. Nε-(carboxymethyl) lysine (CML) and Nε-(carboxyethyl) lysine (CEL) are two typical AGEs, and their contents are often used as indicators of AGEs formation in food. The main methods for detecting AGEs at present include enzyme-linked immunoassay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FL), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (HPLC-MS / MS). Although ELISA is simple, it lacks selectivity and non-specificity, which may result in false positives or overestimation of experimental results. In the analysis of AGEs content in food, HPLC-FL usually uses derivatization reagents such as o-phthaldehyde or fluorenylmethoxycarbonyl chloride (FMOC-Cl) for pre-column derivatization, but the qualitative accuracy and quantitative sensitivity are limited. GC-MS has high sensitivity and qualitative accuracy, but it also needs to use derivatization reaction before quantitative analysis. HPLC-MS / MS has higher instrument sensitivity and more accurate qualitative analysis, but due to the strong polarity and water solubility of CML and CEL, direct sampling analysis has strong matrix effect and low chromatographic resolution. After derivatization of CML and CEL in baked foods using FMOC-Cl, HPLC-MS / MS analysis can effectively reduce the matrix effect. However, due to the complexity of food matrix, there are many macromolecular compounds such as proteins, and the polarity of AGEs is strong and the content is low. In order to realize the selective and efficient enrichment of AGEs in baked foods, solid phase extraction method is usually used for sample purification. Commercial solid phase extraction columns have limited specific surface area and adsorption capacity, poor adsorption selectivity, high cost, and complex operation process. Therefore, it is urgent to develop an enrichment method for advanced glycosylation end products. SUMMARY

[0003] The application aims to provide an enrichment method of advanced glycation end products, which can solve the problems of limited adsorption capacity and poor adsorption selectivity performance when using commercial solid-phase extraction columns to enrich advanced glycation end products.

[0004] The application also aims to provide a detection method of advanced glycation end products.

[0005] To achieve the above-mentioned purposes, the application provides an enrichment method of advanced glycation end products, which adopts the following technical scheme:

[0006] The application provides an enrichment method of advanced glycation end products, which comprises the following steps: mixing a liquid sample containing advanced glycation end products with a magnetic solid-phase extraction agent, performing solid-phase extraction, then performing solid-liquid separation, and then using an eluent to elute the solid obtained by the solid-liquid separation to obtain an enriched liquid containing advanced glycation end products, and drying to obtain an enrichment product; the magnetic solid-phase extraction agent comprises magnetic nanoparticles and a covalent organic framework material modified on the surface of the magnetic nanoparticles, and the covalent organic framework material is mainly prepared by Schiff base condensation reaction of 2,4,6-trihydroxy-1,3,5-benzene tricarboxaldehyde and 4,4"-diamino triphenyl.

[0007] The application provides an enrichment method of advanced glycation end products, which adopts a magnetic solid-phase extraction agent to perform magnetic solid-phase extraction on the advanced glycation end products in the liquid sample containing advanced glycation end products, can realize selective and efficient enrichment of advanced glycation end products, and has the advantages of simple operation, short extraction time, low solvent consumption, and the like, and can overcome the problems of high cost of solid-phase extraction columns and complex operation process of passing through the columns.

[0008] Preferably, the magnetic nanoparticles are amino-functionalized Fe3O4 magnetic nanoparticles. Preferably, the average particle size of the amino-functionalized Fe3O4 magnetic nanoparticles is 50-100 nm. For example, the average particle size of the amino-functionalized Fe3O4 magnetic nanoparticles is 50 nm.

[0009] Preferably, the amino-functionalized Fe3O4 magnetic nanoparticles are prepared by a method comprising the following steps: reacting iron salt, acetate and diamine compound in an alcohol solvent at not less than 200℃ for not less than 6h, solid-liquid separation, washing, and drying to obtain the amino-functionalized Fe3O4 magnetic nanoparticles. Preferably, the iron salt is ferric chloride and / or hydrate of ferric chloride. Preferably, the acetate is alkali metal acetate. Preferably, the diamine compound is one or any combination of butanediamine, pentanediamine, and hexanediamine. Preferably, the hexanediamine is 1,6-hexanediamine. Preferably, the alcohol solvent is a binary alcohol solvent. Preferably, the binary alcohol solvent is selected from one or any combination of ethylene glycol, propylene glycol, and butylene glycol.

[0010] Preferably, the magnetic solid-phase extraction agent is prepared by a method comprising the following steps:

[0011] (1) mixing and reacting 2,4,6-trihydroxy-1,3,5-benzene tricarboxaldehyde and magnetic nanoparticles in a solvent;

[0012] (2) adding 4,4"-diamino triphenyl to the system after the reaction in step (1), continuing the mixing and reaction, solid-liquid separation, and washing to obtain the magnetic solid-phase extraction agent.

[0013] In the preparation of the magnetic solid-phase extraction agent, first mixing and reacting 2,4,6-trihydroxy-1,3,5-benzene tricarboxaldehyde and magnetic nanoparticles can make the aldehyde groups in the 2,4,6-trihydroxy-1,3,5-benzene tricarboxaldehyde react with the amino groups on the surface of the magnetic nanoparticles and bind to the magnetic nanoparticles, and then react with 4,4"-diamino triphenyl to obtain the covalent organic framework material modified on the surface of the magnetic nanoparticles.

[0014] Preferably, in step (1), the mass of the magnetic nanoparticles used corresponds to 0.8-0.105g per 0.3mmol of 2,4,6-trihydroxy-1,3,5-benzene tricarboxaldehyde.

[0015] Preferably, in step (1), the volume of the solvent used corresponds to 10-40mL per 0.3mmol of 2,4,6-trihydroxy-1,3,5-benzene tricarboxaldehyde.

[0016] Preferably, in step (1), the solvent is an alcohol solvent. For example, the solvent is ethanol.

[0017] Preferably, in step (1), the temperature of the mixing and reaction is 50-80℃.

[0018] Preferably, in step (1), the mixing reaction method includes the following steps: mixing 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde and magnetic nanoparticles in a solvent for no less than 30 min, and then letting it stand for no less than 30 min.

[0019] Preferably, the molar ratio of 2,4,6-trihydroxy-1,3,5-benzyltricarboxaldehyde and 4,4”-diaminoterphenyl is 3:(1-4.5).

[0020] Preferably, in step (2), 4,4”-diaminoterphenyl is added in the form of a 4,4”-diaminoterphenyl solution. Preferably, in step (2), the 4,4”-diaminoterphenyl solution is mainly composed of 4,4”-diaminoterphenyl and an alcohol solvent. For example, the alcohol solvent is ethanol.

[0021] Preferably, in step (2), the 4,4”-diaminoterphenyl solution is added dropwise to the system after the reaction in step (1). Preferably, in step (2), the dropwise addition time for each xL of 4,4”-diaminoterphenyl solution is not less than 15 min, and each xL of 4,4”-diaminoterphenyl solution contains 0.45 mmol of 4,4”-diaminoterphenyl. Adding the 4,4”-diaminoterphenyl solution dropwise facilitates better encapsulation of the covalent organic framework material on the surface of the magnetic nanoparticles.

[0022] Preferably, in step (2), the volume of alcohol solvent used for each 0.45 mmol of 4,4”-diaminoterphenyl is 10 to 40 mL.

[0023] Preferably, in step (2), the temperature of the mixing reaction is room temperature. Preferably, in step (2), the time of the mixing reaction is not less than 12.5 hours.

[0024] Preferably, in step (2), the washing process involves soaking the solid obtained from solid-liquid separation in an organic solvent. Preferably, the organic solvent is selected from one or any combination of N,N-dimethylformamide, ethanol, and methanol.

[0025] Preferably, the soaking treatment method includes the following steps: the solid obtained from solid-liquid separation is first soaked in N,N dimethylformamide for no less than 6 hours, then soaked again in N,N dimethylformamide for no less than 12 hours, then soaked in ethanol for no less than 12 hours, and finally soaked in methanol for no less than 8 hours.

[0026] Preferably, the preparation method of the magnetic solid phase extractant further includes the following steps: after washing, drying is performed to obtain the magnetic solid phase extractant.

[0027] Preferably, the enrichment method for advanced glycosylation products further includes the following steps: before eluting the solid obtained from solid-liquid separation with an eluent, the solid obtained from solid-liquid separation is first washed with water and dichloromethane in sequence, and then the washed solid is eluted with an eluent.

[0028] Preferably, the mass of magnetic solid-phase extractant corresponding to each 3 mL liquid sample containing advanced glycation end products is 20–100 mg.

[0029] Preferably, when washing the solid obtained from solid-liquid separation with water and then dichloromethane sequentially, the volume of water corresponding to each 20 mg of magnetic solid-phase extractant is 3 to 10 mL. For example, when washing the solid obtained from solid-liquid separation with water and then dichloromethane sequentially, the volume of water corresponding to each 20 mg of magnetic solid-phase extractant is 5 mL.

[0030] Preferably, when washing the solid obtained from solid-liquid separation with water and then with dichloromethane in sequence, the volume of dichloromethane corresponding to each 20 mg of magnetic solid-phase extractant is 3 to 10 mL. For example, when washing the solid obtained from solid-liquid separation with water and then with dichloromethane in sequence, the volume of dichloromethane corresponding to each 20 mg of magnetic solid-phase extractant is 5 mL.

[0031] Preferably, when washing the solid obtained from solid-liquid separation with water and dichloromethane sequentially, the volume of eluent corresponding to each 20 mg of magnetic solid-phase extractant is 3–10 mL. For example, when washing the solid obtained from solid-liquid separation with water and dichloromethane sequentially, the volume of eluent corresponding to each 20 mg of magnetic solid-phase extractant is 5 mL.

[0032] Preferably, the eluent is mainly prepared by mixing ammonia and methanol. Preferably, the mass fraction of the ammonia is 1-10%. Preferably, the mass ratio of the ammonia to methanol is (2-5):(95-98).

[0033] Preferably, the advanced glycation end products (AGEs) in the liquid sample containing AGEs are carboxymethyl lysine and / or carboxyethyl lysine.

[0034] Preferably, the liquid sample containing advanced glycation end products (AGEs) is obtained from baked goods through pretreatment; the pretreatment method includes the following steps: sequentially subjecting the baked goods to defatting, reduction, hydrolysis, and derivatization treatments. The baked goods include starch-based baked goods and nut-based baked goods, wherein the nut-based baked goods are selected from one or any combination of walnuts, cashews, almonds, hazelnuts, pistachios, sunflower seeds, almonds, peanuts, melon seeds, pine nuts, lotus seeds, macadamia nuts, pecans, chestnuts, and ginkgo nuts.

[0035] Preferably, the defatting process involves centrifuging the baked goods and n-hexane before drying. Preferably, the centrifugation speed is not less than 10,000 r / min. Preferably, the centrifugation is performed at a temperature not lower than 4°C. Preferably, the centrifugation time is not less than 3 min. The centrifugation step can be performed once or repeated multiple times. To improve the defatting effect, preferably, the centrifugation step is repeated multiple times. For example, the centrifugation step is repeated 4 times before drying. Preferably, for each centrifugation, the volume of n-hexane used is 5-10 mL for every 40-100 mg of baked goods.

[0036] Preferably, the reduction treatment method includes the following steps: mixing the defatted baked goods, sodium borate solution, and 1-octanol, then adding sodium borohydride solution, and reacting at a temperature not lower than 4°C. Preferably, the volume of sodium borate solution used for every 40-100 mg of baked goods is 1-3 mL. Preferably, the volume of 1-octanol used for every 40-100 mg of baked goods is 1-3 drops. Preferably, the pH of the sodium borate solution is 9.0-9.2. Preferably, the concentration of the sodium borate solution is 0.2-0.4 mol / L. Preferably, the volume of sodium borohydride solution used for every 40-100 mg of baked goods is 1-2 mL.

[0037] Preferably, the hydrolysis treatment method includes the following steps: mixing the reduced system with hydrochloric acid, keeping it at a temperature not lower than 110°C for not less than 24 hours under an inert atmosphere, separating the solid and liquid, then diluting the liquid obtained from the solid-liquid separation to obtain a diluted solution, and then adjusting the pH of the diluted solution to 8-9. Preferably, the volume of hydrochloric acid used for every 40-100 mg of baked goods is 2-5 mL. Preferably, the concentration of the hydrochloric acid is 10-12 mol / L. Preferably, the volume of the diluted solution for every 40-100 mg of baked goods is 10-12 mL.

[0038] Preferably, the derivatization method includes the following steps: mixing the liquid obtained from hydrolysis with acetonitrile, then adding a borate buffer solution and a derivatizing reagent, allowing it to stand for at least 10 minutes, then adding formic acid, mixing well, and then diluting with acetonitrile solution to obtain a liquid sample containing advanced glycation end products (AGEs). Preferably, the derivatizing reagent is an acetonitrile solution of fluorenemethyloxycarbonyl chloride. Preferably, the concentration of the acetonitrile solution of fluorenemethyloxycarbonyl chloride is 12–15 g / L. Preferably, the pH of the borate buffer solution is 9–9.5. Preferably, the volume ratio of acetonitrile, borate buffer solution, derivatizing reagent, and formic acid used in the derivatization method is 500:200:200:5. Preferably, the mass fraction of the acetonitrile solution is 45–50%. Preferably, in the derivatization method, the volume of the liquid sample containing AGEs corresponding to 500 μL of acetonitrile is 2.5–3 mL.

[0039] When the enriched product obtained by the enrichment method for advanced glycosylation products is analyzed by HPLC-MS / MS, the derivatization method further includes the following steps: before mixing the liquid obtained by hydrolysis and acetonitrile, the liquid obtained by hydrolysis and the mixed internal standard solution are mixed to obtain a mixture, then the mixture is mixed with acetonitrile, and then the borate buffer solution and derivatization reagent are added. Preferably, the volume ratio of the liquid obtained by hydrolysis to the mixed internal standard solution is 10:(0.8-1). Preferably, the volume ratio of the mixture to acetonitrile is 1:(0.8-1). Preferably, the mixed internal standard solution is composed of water and an internal standard; the internal standard is carboxymethyl lysine-d4 and / or carboxyethyl lysine-d4. Preferably, the internal standard is carboxymethyl lysine-d4 and carboxyethyl lysine-d4. Preferably, in the mixed internal standard solution, the concentration of carboxymethyl lysine-d4 is 1.0–1.2 μg / mL; the concentration of carboxyethyl lysine-d4 is 1.0–1.2 μg / mL.

[0040] The technical solution adopted in the detection method of advanced glycation end products of the present invention is as follows:

[0041] A method for detecting advanced glycation end products (AGEs) includes the following steps: analyzing the enriched product obtained by the enrichment method for AGEs as described above using HPLC-MS / MS.

[0042] The method for detecting advanced glycosylation products of the present invention is simple to operate, fast and efficient, low in detection cost, and has high detection accuracy and stability. Attached Figure Description

[0043] Figure 1 This is a schematic flowchart of the detection method for advanced glycosylation products according to Example 2 of the present invention.

[0044] Figure 2 This is a schematic diagram showing the adsorption results of different magnetic solid phase extractants on CML and CEL in Experiment Example 2 of the present invention. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0046] I. Specific embodiments of the method for enriching advanced glycosylation products of the present invention are as follows:

[0047] Example 1

[0048] The enrichment method for advanced glycosylation products in this embodiment specifically includes the following steps:

[0049] (1) Pretreatment of samples containing advanced glycation end products

[0050] S1. Grind the baked food sample (the baked food sample is a biscuit, named Cookie 1) to a particle size of not less than 40 mesh. Accurately weigh 40 mg of the ground baked food sample and place it in a 50 mL centrifuge tube. Add 5 mL of n-hexane, vortex thoroughly, and centrifuge at 4℃ and 10000 r / min for 3 min. Discard the n-hexane layer. Then repeat the above n-hexane defatting process 3 times. Finally, use nitrogen gas at 40℃ to blow the solid obtained by centrifugation to remove n-hexane and obtain a defatted sample.

[0051] S2. Transfer the defatted sample to a hydrolysis tube, add 1.5 mL of sodium borate solution (0.2 mol / L, pH = 9.2), and add 1 drop of 1-octanol. Then, slowly add 1 mL of sodium borohydride solution (sodium borohydride solution is prepared by mixing 0.1 mol / L sodium borohydride aqueous solution and 0.1 mol / L NaOH aqueous solution in a volume ratio of 1:1). Stir overnight at 4°C to carry out reduction treatment and obtain a reduced solution. Then, add 2.6 mL of 12 mol / L hydrochloric acid dropwise to the reduced solution. Screw the cap on the hydrolysis tube, evacuate and purge with nitrogen three times, tighten the stopcock, and incubate the sample at 110°C for 24 h to carry out the hydrolysis reaction. Then, filter the system after the hydrolysis reaction, pour the filtrate into a volumetric flask, and dilute to 10 mL with water. Then, adjust the pH of the liquid in the volumetric flask to 8-9 with 50% potassium hydroxide solution to obtain the hydrolysis dilution.

[0052] S3. Mix 1 mL of hydrolysis diluent and 100 μL of mixed internal standard solution (the mixed internal standard solution consists of water, carboxymethyl lysine-d4, and carboxyethyl lysine-d4, with a concentration of 1.0 μg / mL for both). Then, vortex 500 μL of the mixture and 500 μL of acetonitrile. Next, add 200 μL of borate buffer solution (pH = 9) and 200 μL of 15 g / L fluorenemethyloxycarbonyl chloride acetonitrile solution to the vortexed liquid, vortex, and let stand for 10 min for derivatization. Then, add 5 μL of formic acid, mix, and dilute to 3 mL with 50% acetonitrile solution. Vortex to obtain the sample processing solution, i.e., the liquid sample containing advanced glycation end products (AGEs).

[0053] (2) Enrichment of advanced glycation end products

[0054] Place the sample solution obtained in step (1) into a 50 mL centrifuge tube, then add 20 mg of magnetic solid phase extractant, vortex for 20 min, then place a magnet on the outer wall of one side of the centrifuge tube to separate the magnetic solid phase extractant from the solution, pour out the solution, remove the magnet, then add 5 mL of water and 5 mL of dichloromethane to the centrifuge tube to wash the magnetic solid phase extractant. After each wash, use an external magnetic field to achieve phase separation, then add 5 mL of eluent (the eluent is prepared by mixing 10% ammonia water and methanol in a volume ratio of 5:95) to the centrifuge tube for elution, and then concentrate the eluent to near dryness using nitrogen gas at 40 °C to obtain the enriched product;

[0055] The magnetic solid-phase extractant was prepared by the following method:

[0056] ① Place 0.30 mmol of 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde (Tp) into a 250 mL three-necked flask, add 10 mL of anhydrous ethanol, and sonicate until the 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde is completely dissolved; then add 0.1050 g of amino-functionalized Fe3O4 magnetic nanoparticles to the three-necked flask, and continue sonicating for 10 min to disperse the magnetic nanoparticles evenly. Then, mechanically stir the material in the three-necked flask at 50 °C for 30 min, and then let it stand at 50 °C for 30 min to obtain a dispersion; the preparation method of the amino-functionalized Fe3O4 magnetic nanoparticles is as follows: place 4.0 g of FeCl3·6H2O and 120 mL of ethylene glycol into In a 500 mL three-necked flask, FeCl3·6H2O was dissolved by mechanical stirring in a 50 °C water bath. Then, 8.0 g of anhydrous sodium acetate and 26.0 g of 1,6-hexanediamine were added to the three-necked flask, and the mixture was stirred vigorously for 0.5 h to obtain a homogeneous solution. The homogeneous solution was then transferred to a hydrothermal reactor and reacted at 200 °C for 6 h. The reaction mixture was then transferred to a beaker and separated using a magnet. The supernatant was discarded, and the resulting solid was washed five times with deionized water and 95% ethanol, vortexing for 5 min each time. Finally, the washed solid was placed in a fume hood to air dry the solvent and then dried in a vacuum drying oven at 50 °C for 12 h to obtain amino-functionalized Fe3O4 magnetic nanoparticles with a particle size of 50–100 nm.

[0057] ② Weigh 0.45 mmol of 4,4”-diaminoterphenyl (Tph), put it into a small beaker, and then add 40 mL of anhydrous ethanol to dissolve the 4,4”-diaminoterphenyl to obtain an ethanol solution of 4,4”-diaminoterphenyl.

[0058] ③ Under normal temperature and stirring conditions, the ethanol solution of 4,4”-diaminoterphenyl from step ② was slowly added dropwise to the dispersion from step ① over a period of 15 min. After the addition was complete, the reaction was stirred for another 30 min. Then the stirring speed was reduced and the reaction was continued for another 12 h. The reaction mixture was then transferred to a 250 mL round-bottom flask and rotary evaporated at 45 °C to remove the ethanol. The solid was then soaked in 50 mL of N,N dimethylformamide (DMF) for 6 h, filtered, and then soaked in DMF for 12 h. The solid was then filtered again and soaked in 95% ethanol for 12 h. The solid was then filtered again and soaked in methanol for 12 h. After filtration, the solid was soaked in methanol for 8 h, filtered again, and then air-dried in a fume hood for 8 h. Finally, the solid was vacuum-dried at 50 °C for 12 h to obtain the magnetic solid-phase extractant.

[0059] Comparative Example 1

[0060] The difference between the enrichment method for advanced glycation end products in this comparative example and the enrichment method for advanced glycation end products in Example 1 is that the magnetic solid phase extractant used in the enrichment method for advanced glycation end products in this comparative example is prepared by replacing 4,4”-diaminoterphenyl (Tph) with p-phenylenediamine (Pa-1).

[0061] Comparative Example 2

[0062] The only difference between the enrichment method of advanced glycation end products in this comparative example and the enrichment method of advanced glycation end products in Comparative Example 1 is that the magnetic solid phase extractant used in the enrichment method of advanced glycation end products in this comparative example is prepared by replacing p-phenylenediamine with benzidine (BD).

[0063] II. Specific embodiments of the detection method for advanced glycation end products of the present invention are as follows:

[0064] Example 2

[0065] The method for detecting advanced glycation end products in this embodiment, such as... Figure 1 As shown, a magnetic solid-phase extractant was first synthesized, and the sample containing advanced glycation end products (AGEs) was pretreated to obtain a liquid sample containing AGEs. The liquid sample was then subjected to solid-phase extraction using the magnetic solid-phase extractant, followed by magnetic separation and desorption using an eluent to obtain a concentrated solution, which was then analyzed by HPLC-MS / MS. The methods for synthesizing the magnetic solid-phase extractant, pretreatment, solid-phase extraction, and desorption were performed according to the method in Example 1; specifically, the following steps were included:

[0066] The enriched product obtained in Example 1 was dissolved in acetonitrile solution with a mass fraction of 50% to obtain a 1 mL test solution. The test solution was then analyzed by HPLC-MS / MS (repeated 3 times). The concentration of each target compound in the baked sample was determined according to the standard curve of each target compound (carboxymethyl lysine and carboxyethyl lysine). The test results showed that the concentrations of carboxymethyl lysine and carboxyethyl lysine in the baked sample of Example 1 were 40.50±0.71 mg / kg and 11.78±0.54 mg / kg, respectively.

[0067] The standard curves for each target compound (carboxymethyl lysine and carboxyethyl lysine) were established as follows:

[0068] (1) Prepare a stock solution of 1 mg / mL of carboxymethyl lysine standard with water and prepare a stock solution of 0.5 mg / mL of carboxyethyl lysine standard with water. Then mix the two stock solutions to prepare a mixed standard solution. The concentrations of carboxymethyl lysine and carboxyethyl lysine in the mixed standard solution are both 10 μg / mL.

[0069] Carboxymethyl lysine-d4 and carboxyethyl lysine-d4 internal standards were prepared into stock solutions of 1 mg / mL, and then mixed internal standard solutions were prepared. The concentrations of carboxymethyl lysine-d4 and carboxyethyl lysine-d4 in the mixed internal standard solutions were both 1 μg / mL.

[0070] (2) Accurately pipette a certain volume (5μL, 10μL, 20μL, 40μL, 80μL) of mixed standard solution, then add 100μL of mixed internal standard solution, and dilute with water to 1mL to obtain standard solutions of different concentrations;

[0071] (3) Pretreatment of standard solutions of different concentrations was performed according to step (1) of the enrichment method for advanced glycosylation products in Example 1, followed by enrichment according to step (2) of the enrichment method for advanced glycosylation products in Example 1 to obtain a concentrate. The concentrate was then dissolved in acetonitrile solution with a mass fraction of 50% to obtain a 1 mL test solution. HPLC-MS / MS analysis was then performed to establish standard curves for each target compound. The standard curve for carboxymethyl lysine was: y = 0.0193x + 0. The correlation coefficient R = 0.9985, and the standard curve for carboxyethyl lysine is: y = 0.0147x + 0.0176, with a correlation coefficient R = 0.9983. The limits of detection (LOD) and quantitation (LOQ) for carboxymethyl lysine are 0.65 ng / mL and 2.15 ng / mL, respectively; the limits of detection (LOD) and quantitation (LOQ) for carboxyethyl lysine are 0.35 ng / mL and 1.16 ng / mL, respectively.

[0072] The chromatographic column used for HPLC-MS / MS analysis was a Waters C18 (100 mm × 2.1 mm) column. The column temperature was 35℃; mobile phase A was a 0.5% formic acid aqueous solution, and mobile phase B was acetonitrile; gradient elution was used, with the following conditions: 0-5 min, 50% A-41% A; 5.1-9.0 min, 41% A-5% A; 9.1-11 min, 5% A-50% A; injection volume was 10 μL; flow rate was 300 μL / min; the mass spectrometry conditions for HPLC-MS / MS analysis were as follows: ion source: electrospray ionization source (ESI); scanning mode: positive ion scan; detection mode: multiple reaction monitoring (MRM); electrospray voltage: 5500 V; curtain gas pressure: 30 psi; auxiliary gas 1 pressure: 70 psi; auxiliary gas 2 pressure: 70 psi; ion source temperature: 500℃; the quantitative ion pairs, qualitative ion pairs, residence time, collision energy (CE), and declustering voltage (DP) of each compound are shown in Table 1.

[0073] Table 1. Quantitative ion pairs, qualitative ion pairs, residence time, collision energy (CE), and declustering voltage (DP) for each compound.

[0074]

[0075] Experimental Example 1

[0076] Advanced glycation end products (AGEs) were enriched in different commercially available baked goods samples (including 15 types of biscuits and 15 types of nuts) using the enrichment method for AGEs described in Example 1. Then, the AGEs were detected using the detection method described in Example 2 (each sample was tested three times) to obtain the concentrations of carboxymethyl lysine and carboxyethyl lysine in the commercially available baked goods samples. The results are shown in Table 2. The 15 types of biscuits were: Cookie 1, Cookie 2, Wafer Biscuits, Milk Biscuits, Waffles, Rice Biscuits, Shortbread Biscuits, Steamed Buns, Sachima, Swiss Rolls, Whole Wheat Bread, Tear-Apart Bread, Whole Wheat Toast, Cheesecake, and Sandwich Cake. The 15 types of nuts were: Walnuts, Cashews, Almonds, Hazelnuts, Pistachios, Sunflower Seeds, Almonds, Peanuts, Melon Seeds, Pine Nuts, Lotus Seeds, Macadamia Nuts, Pecans, Cheesecake, and Ginkgo Nuts.

[0077] Table 2. Concentrations of carboxymethyl lysine and carboxyethyl lysine in commercially available baked goods samples.

[0078]

[0079] Experimental Example 2

[0080] To evaluate the solid-phase extraction effect of different magnetic solid-phase extractants on advanced glycation end products (AGEs), equal amounts of magnetic solid-phase extractants (named TpTph, TpPa-1, and TpBD, respectively) with the same particle size from Examples 1 and Comparative Examples 1-2 were placed in the same solution containing carboxymethyl lysine (CML) or carboxyethyl lysine (CEL) for adsorption tests. After the same adsorption test time, elution was performed under the same conditions, and the concentration of carboxymethyl lysine or carboxyethyl lysine in the eluent was measured. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the magnetic solid phase extractant in Example 1 has a better adsorption effect on advanced glycation end products (CML and CEL) than the magnetic solid phase extractants in Comparative Examples 1-2.

[0081] Experimental Example 3

[0082] Six parallel measurements were performed on the same baked food sample within one day, and also over five days. The relative standard deviation (RSD) of the results represents the intra-day precision and inter-day precision, respectively. Spiking recovery experiments were also conducted at three different spiking levels (low, medium, and high) (each sample was tested three times at the same spiking level). The results are shown in Table 3. In this experimental example, "biscuit" refers to wafer biscuits, and "nuts" refers to walnuts.

[0083] Table 3. Test results of spiked recovery, intra-day precision, and inter-day precision.

[0084]

[0085] Experiment Example 4

[0086] This experimental example evaluates the selective adsorption effect of the magnetic solid-phase extractant used in Example 1 on advanced glycation end products by measuring the matrix effect; the specific method is as follows.

[0087] A standard working solution containing carboxymethyl lysine, carboxyethyl lysine, carboxymethyl lysine-d4, and carboxyethyl lysine-d4 was prepared using water (the concentrations of carboxymethyl lysine, carboxyethyl lysine, carboxymethyl lysine-d4, and carboxyethyl lysine-d4 in the standard working solution were equal, ranging from 25 to 400 ng / mL). Then, 500 μL of the standard working solution and 500 μL of acetonitrile were vortexed together. Finally, 200 μL of borate buffer solution (pH = 9) and 200 μL of a 1% concentration solution were added to the vortexed liquid. A 5 g / L fluorene methoxycarbonyl chloride solution in acetonitrile was vortexed and allowed to stand for 10 min for derivatization. Then, 5 μL of formic acid was added and mixed. The solution was then diluted to 3 mL with 50% acetonitrile solution and vortexed to obtain the standard sample treatment solution. The solution was then analyzed by HPLC-MS / MS using the method described in Example 2. The peak area ratio of carboxymethyl lysine, carboxyethyl lysine and the corresponding deuterated internal standard was linearly regressed against the concentration ratio to obtain the standard working curves of carboxymethyl lysine and carboxyethyl lysine, which were named Curve 1 and Curve 2, respectively.

[0088] Meanwhile, biscuits and nuts from baked goods were selected as typical matrices. The biscuits and nuts were pretreated according to step (1) of the enrichment method for advanced glycation end products in Example 1 to obtain corresponding sample treatment solutions. Each sample treatment solution was then placed in a 50 mL centrifuge tube, and 20 mg of magnetic solid-phase extractant (the same magnetic solid-phase extractant used in Example 1) was added. The tubes were vortexed for 20 min, and a magnet was placed on the outer wall of one side of the centrifuge tube to separate the magnetic solid-phase extractant from the solution. Carboxymethyl lysine, carboxyethyl lysine, carboxymethyl lysine-d4, and carboxyethyl lysine-d4 were added to the separated liquid to obtain a matrix standard working solution (the concentrations of carboxymethyl lysine, carboxyethyl lysine, carboxymethyl lysine-d4, and carboxyethyl lysine-d4 in the matrix standard working solution were equal, ranging from 25 to 400 ng / mL). Then, a 500 μL volume of the matrix standard working solution and the volume of the matrix standard working solution were added to the centrifuge tube. 500 μL of acetonitrile was vortexed and mixed. Then, 200 μL of borate buffer solution (pH=9) and 200 μL of 15 g / L fluorene methoxycarbonyl chloride acetonitrile solution were added to the vortexed liquid, vortexed and mixed, and allowed to stand for 10 min for derivatization. Then, 5 μL of formic acid was added and mixed. The solution was then diluted to 3 mL with 50% acetonitrile solution and vortexed to obtain the standard sample treatment solution. HPLC-MS / MS analysis was performed using the method in Example 2. The peak area ratio of carboxymethyl lysine, carboxyethyl lysine and the corresponding deuterated internal standard was linearly regressed against the concentration ratio to obtain the standard working curves of carboxymethyl lysine and carboxyethyl lysine. The standard working curves of carboxymethyl lysine and carboxyethyl lysine for biscuits were named Curve 3 and Curve 4, respectively, and the standard working curves of carboxymethyl lysine and carboxyethyl lysine for nuts were named Curve 5 and Curve 6, respectively.

[0089] The applicable concentration ranges of carboxymethyl lysine for curves 1, 3 and 5, the applicable concentration ranges of carboxyethyl lysine for curves 2, 4 and 6, the equations of each curve and their correlation coefficients, and the slope ratios of curves 3 and 1 (I), 5 and 1 (II), 4 and 2 (III), and 6 and 2 (IV) are shown in Table 4.

[0090] Table 4 shows the applicable concentration ranges of carboxymethyl lysine and carboxyethyl lysine for each standard working curve, the equations of each curve, their correlation coefficients, and the slope ratios I, II, III, and IV.

[0091]

[0092] As shown in Table 4, the slope ratios of curves 3 and 1 (I), 5 and 1 (II), 4 and 2 (III), and 6 and 2 (IV) are 1.10, 1.04, 1.08, and 1.15, respectively. This indicates that the differences in slope between curves 3 and 1, 5 and 1, 4 and 2, and 6 and 2 are all less than 10%, proving that the magnetic solid-phase extractant in Example 1 can effectively purify the baked goods matrix, reduce the matrix effect, and has good selective adsorption for advanced glycation end products.

Claims

1. A method for enriching advanced glycosylation products, characterized in that, Includes the following steps: A liquid sample containing advanced glycation end products (AGEs) was mixed with a magnetic solid-phase extractant for solid-phase extraction, followed by solid-liquid separation. The solid obtained from the solid-liquid separation was then eluted with an eluent to obtain a concentrated solution containing AGEs. After drying, the concentrated product was obtained. The magnetic solid-phase extractant comprises magnetic nanoparticles and a covalent organic framework material modified on the surface of the magnetic nanoparticles. The covalent organic framework material is prepared by Schiff base condensation reaction of 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde and 4,4''-diaminoterphenyl. The magnetic nanoparticles are amino-functionalized Fe3O4 magnetic nanoparticles. The AGEs in the liquid sample containing AGEs are carboxymethyl lysine and / or carboxyethyl lysine.

2. The method for enriching advanced glycosylation products as described in claim 1, characterized in that, The average particle size of the amino-functionalized Fe3O4 magnetic nanoparticles is 50~100 nm.

3. The method for enriching advanced glycosylation products as described in claim 2, characterized in that, The amino-functionalized Fe3O4 magnetic nanoparticles are prepared by a method comprising the following steps: reacting iron salt, acetate and diamine compound in an alcohol solvent at a temperature of not less than 200°C for not less than 6 hours, followed by solid-liquid separation, washing, and drying to obtain amino-functionalized Fe3O4 magnetic nanoparticles.

4. The method for enriching advanced glycosylation products as described in claim 3, characterized in that, The iron salt is ferric chloride and / or ferric chloride hydrate; the acetate is an alkali metal acetate; the diamine compound is one or any combination of butanediamine, pentanediamine, and hexanediamine; the alcohol solvent is a diol solvent; the diol solvent is selected from one or any combination of ethylene glycol, propylene glycol, and butanediol.

5. The method for enriching advanced glycosylation products as described in any one of claims 2-4, characterized in that, The magnetic solid-phase extractant is prepared by a method comprising the following steps: (1) 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde and magnetic nanoparticles were mixed and reacted in a solvent; (2) Add 4,4''-diaminoterphenyl to the system after the reaction in step (1), continue to mix and react, separate the solid and liquid, and wash to obtain the magnetic solid phase extractant.

6. The method for enriching advanced glycosylation products as described in claim 5, characterized in that, In step (1), the temperature of the mixing reaction is 50~80℃.

7. The method for enriching advanced glycosylation products as described in claim 5, characterized in that, In step (1), the mixing reaction method includes the following steps: mixing 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde and magnetic nanoparticles in a solvent for no less than 30 min, and then letting it stand for no less than 30 min.

8. The method for enriching advanced glycosylation products as described in claim 5, characterized in that, In step (2), 4,4''-diaminoterphenyl is added in the form of a 4,4''-diaminoterphenyl solution; the 4,4''-diaminoterphenyl solution is composed of 4,4''-diaminoterphenyl and an alcohol solvent.

9. The method for enriching advanced glycosylation products as described in claim 5, characterized in that, In step (2), the temperature of the mixing reaction is room temperature; the time of the mixing reaction is not less than 12.5 hours.

10. The method for enriching advanced glycosylation products as described in claim 1, characterized in that, The eluent is prepared by mixing ammonia and methanol.

11. The method for enriching advanced glycosylation products as described in claim 10, characterized in that, The mass fraction of the ammonia water is 1~10%; the mass ratio of the ammonia water to the methanol is (2~5):(95~98).

12. The method for enriching advanced glycosylation products as described in claim 1, characterized in that, The liquid sample containing advanced glycation end products was prepared from baked goods through pretreatment. The pretreatment method includes the following steps: sequentially subjecting the baked food to defatting, reduction, hydrolysis and derivatization treatments.

13. A method for detecting advanced glycation end products (AGEs), characterized in that, Includes the following steps: The late-stage glycosylation end products were enriched using the enrichment method described in any one of claims 1-12, and then analyzed by HPLC-MS / MS.

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