A rapid, high-throughput method for the quantitative determination of glucose and fructose in food

Abstract

The application discloses a rapid and high-throughput quantitative detection method for glucose and fructose in food. The method comprises the following steps: using a compound TMNTA as a reactive matrix to perform a derivatization reaction with glucose and fructose, and performing qualitative and quantitative analysis on the target by detecting the derivatization product through MALDI-TOF MS and MALDI-TOF / TOF MS / MS. The quantitative detection method is simple and rapid in operation, and the entire experimental process can be completed within 2 minutes; the quantitative result is accurate, and the rapid and high-throughput detection of glucose and fructose in food samples can be met.

Description

Technical field:

[0001] The present invention relates to the technical field of food analysis and detection, and in particular to a rapid and high-throughput quantitative detection method for glucose and fructose in food. Background technology:

[0002] Glucose and fructose are two common and important monosaccharides. They are isomers of each other and are naturally present or added as sweeteners in a variety of foods. They are important nutrients in food and provide energy for human life processes. The content of glucose and fructose is an important quality indicator for certain foods. For example, the "National Food Safety Standard for Honey" (GB14963-2011) stipulates that the sum of the glucose and fructose content in honey should be ≥ 60% (g / 100g). The "Orange Juice and Orange Juice Beverages" (BG / T21731-2008) stipulates that the glucose and fructose content should be within the range of 20.0-35.0g / kg, and the glucose-to-fructose ratio should be ≤ 1.0. Therefore, accurate monitoring of the glucose and fructose content in foods is of great significance for food quality evaluation and control.

[0003] Because glucose and fructose lack favorable UV-absorbing groups in their structures, direct detection using common UV detectors is particularly difficult. Therefore, currently common detection methods include high-performance liquid chromatography (HPLC) coupled with specialized detectors such as differential refractive index detector (RID), evaporative light scattering detector (ELSD), and charged aerosol detector (CAD), ion chromatography, and liquid chromatography-mass spectrometry. These methods all require time-consuming chromatographic separations and are incapable of rapid, high-throughput detection. Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF MS) offers advantages such as accuracy, rapid analysis, high sensitivity, high throughput, and simplified sample preparation, and is expected to play a significant role in the rapid, high-throughput detection of glucose and fructose in foods. However, this technique suffers from matrix signal interference in the low mass-to-charge ratio region, poor mass spectrometric signal reproducibility, and difficulty in quantifying isomers separately, significantly hindering the quantitative determination of glucose and fructose in foods. There is currently no report on the separate quantification of glucose and fructose in food using MALDI-TOF MS technology.

[0004] Achieving "faster detection speed, higher analytical throughput, higher sensitivity, and higher accuracy" is a technical challenge facing traditional food testing technologies. Therefore, there is an urgent need to establish an accurate, simple, rapid, high-throughput, and highly sensitive analytical method for the quantitative detection of glucose and fructose in food. Summary of the invention:

[0005] The present invention solves the problems of time-consuming glucose and fructose detection and low detection throughput in the prior art, and provides a rapid and high-throughput method for quantitative detection of glucose and fructose in food. The quantitative detection method proposed in the present invention is simple and fast to operate, and the entire experimental process can be completed within 2 minutes; the quantitative results are accurate, and can meet the needs of rapid and high-throughput detection of glucose and fructose in food samples.

[0006] The first object of the present invention is to provide a compound named 4-trimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-(3-aminophenylboronic acid) ((4-trimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-tri azine-2-(3-aminophenylboronic acid), TMNTA)), with a structural formula as shown in Formula 1:

[0007]

[0008] The second object of the present invention is to provide a method for preparing the compound, using cyanuric chloride as a raw material molecule, and obtaining compound 3 through a three-step substitution reaction, that is, the compound, whose chemical reaction equation is shown in Formula 2:

[0009]

[0010] Preferably, the preparation method specifically comprises the following steps:

[0011] (1) Synthesis of Compound 1: Cyanuric chloride and 1-methoxynaphthalene were added to a reaction vessel containing toluene, wherein the molar ratio of cyanuric chloride to 1-methoxynaphthalene was 0.5-2.0:1, and the mass volume ratio of cyanuric chloride to toluene was 4-10:100 g / mL. Aluminum chloride was then added in portions, wherein the molar ratio of 1-methoxynaphthalene to aluminum chloride was 0.5-2.0:1. The mixture was stirred at room temperature for 12-20 hours. After the reaction was completed, the reaction was quenched with hydrochloric acid, and the organic phases were extracted and combined, washed, dried in vacuo, and then recrystallized to obtain Compound 1;

[0012] (2) Synthesis of Compound 2: Compound 1 and 3-aminophenylboronic acid were added to a reaction vessel containing acetone, wherein the molar ratio of Compound 1 to 3-aminophenylboronic acid was 0.5-2.0:1, and the mass volume ratio of 3-aminophenylboronic acid to acetone was 1:10-30 g / mL. A sodium hydroxide aqueous solution was added, wherein the molar ratio of 3-aminophenylboronic acid to sodium hydroxide was 0.5-2.0:1. The mixture was stirred at 0°C for 2-4 hours. After the reaction was completed, the reaction solution was poured into ice water, filtered, and vacuum dried to obtain Compound 2;

[0013] (3) Synthesis of Compound 3: Compound 2 and trimethylamine were added to a reaction vessel containing tetrahydrofuran, the mass volume ratio of Compound 2 to tetrahydrofuran being 1:10-30 g / mL, the concentration of trimethylamine in the reaction system being 1 / 5-2 / 3 mol / L, the mixture was stirred at room temperature for 0.5-1.5 h, and the reaction solution was then concentrated in vacuo. The obtained solid was dissolved in tetrahydrofuran and stirred at room temperature for 0.25-0.75 h, filtered, washed with cold tetrahydrofuran, and purified to obtain Compound 3.

[0014] More preferably, the preparation method specifically comprises the following steps:

[0015] (1) Synthesis of Compound 1: Cyanuric chloride and 1-methoxynaphthalene were added to a reaction vessel containing toluene, wherein the molar ratio of cyanuric chloride to 1-methoxynaphthalene was 1:1, and the mass volume ratio of cyanuric chloride to toluene was 7-8:100 g / mL. Aluminum chloride was then added in portions, wherein the molar ratio of 1-methoxynaphthalene to aluminum chloride was 5:6. The mixture was stirred at room temperature for 16 h. After the reaction was completed, the reaction was quenched with hydrochloric acid, and the organic phases were extracted and combined, washed, dried in vacuo, and then recrystallized to obtain Compound 1.

[0016] (2) Synthesis of Compound 2: Compound 1 and 3-aminophenylboronic acid were added to a reaction vessel containing acetone, wherein the molar ratio of Compound 1 to 3-aminophenylboronic acid was 1:1, and the mass volume ratio of 3-aminophenylboronic acid to acetone was 1:18-19 g / mL. A sodium hydroxide aqueous solution was added, wherein the molar ratio of 3-aminophenylboronic acid to sodium hydroxide was 1:1. The mixture was stirred and reacted at 0°C for 3 hours. After the reaction was completed, the reaction solution was poured into ice water, filtered, and vacuum dried to obtain Compound 2;

[0017] (3) Synthesis of Compound 3: Compound 2 and trimethylamine were added to a reaction vessel containing tetrahydrofuran, the mass volume ratio of Compound 2 to tetrahydrofuran being 1:16 g / mL, the concentration of trimethylamine in the reaction system being 0.43 mol / L, the mixture was stirred at room temperature for 1 h, and the reaction solution was then concentrated in vacuo. The obtained solid was dissolved in tetrahydrofuran and stirred at room temperature for 0.5 h, filtered, washed with cold tetrahydrofuran, and purified to obtain Compound 3.

[0018] The third object of the present invention is to provide the use of the compound in the quantitative detection of glucose and fructose in food.

[0019] A fourth object of the present invention is to provide a rapid, high-throughput method for quantitatively detecting glucose and fructose in food, comprising the steps of using the compound as a reactive matrix to undergo a derivatization reaction with glucose and fructose, and detecting the derivatized products by MALDI-TOF MS and MALDI-TOF / TOF MS / MS to perform qualitative and quantitative analysis of the target substances.

[0020] The present invention uses a boronic acid-modified compound as a reactive matrix to undergo a specific derivatization reaction with glucose and fructose. After derivatization, the signal response of the target object is enhanced, and the detection sensitivity is improved. No additional matrix needs to be added after derivatization, which solves the problem of matrix background interference. The use of a stable isotope compound as an internal standard can effectively correct for mass spectrometry signal fluctuations and sample matrix effects, thereby achieving accurate quantitative analysis. The concentration relationship of glucose and fructose is obtained by using the intensity ratio of two daughter ions with different responses of glucose and fructose derivatization products, thereby achieving separate quantitative analysis of glucose and fructose, and solving the problem of quantitative detection of isomers by MALDI mass spectrometry.

[0021] The reaction formula for the derivatization reaction between the compound (TMNTA) and glucose and fructose is shown in Formula 3 below:

[0022]

[0023] Preferably, the quantitative detection method comprises the following steps: dissolving the compound (boronic acid-modified compound TMNTA) in an acetonitrile solution, then vortex mixing with glucose and fructose to perform a (specific) derivatization reaction; after the derivatization reaction, spotting the reaction solution on a target plate, air-drying at room temperature, and then feeding it into a MALDI mass spectrometer to quantitatively detect glucose and fructose using a stable isotope internal standard method; and analyzing the relationship between the glucose and fructose contents using MALDI-TOF / TOF MS / MS.

[0024] Preferably, the MALDI mass spectrometry detection conditions are as follows: using a laser with a wavelength of 250-360 nm, the primary mass spectrometer (MALDI-TOF MS) adopts an acceleration voltage of 19-21 kV, the detection mode is a positive ion reflectron mode, the detection frequency is 800-1200 Hz, and the laser energy is 30%-50%; the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) adopts an initial acceleration voltage of 5-8 kV, a dissociation cell acceleration voltage of 18-20 kV, a detection frequency of 200-500 Hz, the laser energy for parent ion detection is set to 30%-50%, and the laser energy for daughter ion detection is set to 50%-70%.

[0025] Further preferably, the MALDI mass spectrometry detection conditions are as follows: using a laser with a wavelength of 266-337 nm, the primary mass spectrometer (MALDI-TOF MS) adopts an acceleration voltage of 19-21 kV, the detection mode is positive ion reflectron mode, the detection frequency is 800-1200 Hz, and the laser energy is 30%-50%; the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) adopts an initial acceleration voltage of 5-8 kV, a dissociation cell acceleration voltage of 18-20 kV, a detection frequency of 200-500 Hz, the laser energy for parent ion detection is set to 30%-50%, and the laser energy for daughter ion detection is set to 50%-70%.

[0026] Further preferably, each mass spectrum detected by the primary mass spectrometry (MALDI-TOF MS) is the average result of 800-1500 laser scans; each mass spectrum detected by the secondary mass spectrometry (MALDI-TOF / TOF MS / MS) is the average result of 300-1000 laser scans.

[0027] To achieve accurate quantitative analysis, stable isotope internal standardization was used to quantitatively detect glucose and fructose using MALDI mass spectrometry. The relationship between glucose and fructose content was analyzed using secondary mass spectrometry (MALDI-TOF / TOF MS / MS).

[0028] Preferably, when MALDI mass spectrometry is used to quantitatively detect glucose and fructose in actual samples, [ 13 C6] Glucose was used as a stable isotope internal standard to correct the mass spectrometry signal fluctuations of all target compounds and the matrix effects caused by the samples to provide accurate quantitative data.

[0029] Preferably, the quantitative detection method is characterized in that it specifically comprises the following steps:

[0030] S1. Glucose or fructose standard solutions of different concentrations containing a fixed concentration of a stable isotope internal standard are prepared, and derivatized with the compound. The resulting reaction solution containing the standard derivatized product is subjected to MALDI-TOF MS analysis, with the concentration of the glucose or fructose standard solution as the abscissa and the intensity ratio of the mass spectral peak of the standard derivatized product to the stable isotope internal standard derivatized product as the ordinate, to establish standard working curves for glucose and fructose, respectively. The standard working curves for glucose and fructose are added together to obtain a standard working curve containing both glucose and fructose concentrations.

[0031] S2. Glucose and fructose are prepared at the same concentrations, and derivatized with the compounds. The resulting reaction solutions containing glucose-derivative products and fructose-derivative products are mixed in different proportions and analyzed by MALDI-TOF / TOF MS / MS. A working curve for the relationship between glucose and fructose concentrations is established, with the ratio of glucose to fructose concentrations as the abscissa and the intensity ratio of the two differentially responsive product ions as the ordinate.

[0032] S3. Derivatizing the compound with glucose and fructose test samples to which stable isotope internal standards are added, and then subjecting the derivatized products of the test samples to MALDI-TOF MS and MALDI-TOF / TOF MS / MS analysis. Substituting the ratio of the two response difference daughter ions of glucose and fructose into the working curve obtained in step S2, thereby calculating the concentration ratio of glucose and fructose; substituting the mass spectrum peak intensity ratio of the derivatized product of the test sample and the derivatized product of the stable isotope internal standard and the concentration ratio of glucose and fructose into the standard working curve obtained in step S1, thereby calculating the concentrations of glucose and fructose in the test sample.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1. The present invention uses compound TMNTA as a "reactive matrix" to carry out derivatization reaction on glucose and fructose. The principle is that boric acid and cis-dihydroxyl undergo a highly specific binding reaction. After derivatization, the response of the target on the MALDI mass spectrometer is greatly enhanced, and the detection sensitivity is effectively improved. Since no additional MALDI matrix is ​​added, there is no matrix background signal interference (see Figure 3 ); and by introducing stable isotope compounds as internal standards to correct mass spectrometry signal fluctuations and sample matrix effects, accurate quantitative analysis of glucose and fructose can be achieved.

[0035] 2. The present invention uses the two daughter ions of glucose and fructose derivatives with different responses to obtain the concentration relationship of glucose and fructose (see Figure 4 and Figure 5 ), and combined with the additive linear relationship of glucose and fructose, the accurate quantitative analysis of glucose and fructose in the sample was finally achieved, solving the problem that MALDI mass spectrometry could not perform quantitative analysis of isomers separately due to the lack of a chromatographic separation process.

[0036] 3. The quantitative detection method proposed in this invention is quick and easy to operate. After the test sample is derivatized with the "reactive matrix" TMNTA, it is directly subjected to MALDI-TOF MS and MALDI-TOF / TOF MS / MS analysis. No additional MALDI matrix and chromatographic separation steps are required. It has the advantages of simplicity, rapidity, and high throughput, and can realize rapid and high-throughput quantitative analysis of glucose and fructose in food samples. Description of the drawings:

[0037] Figure 1 For the compound TMNTA 1 HNMR spectrum.

[0038] Figure 2 The UV absorption spectra of compounds TMNTA, α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.

[0039] Figure 3 MALDI-TOF MS spectra of blank solvent (A), glucose (B) and fructose (C) after TMNTA derivatization.

[0040] Figure 4 Figure 3 shows the MALDI-TOF / TOF MS / MS mass spectrum of the glucose-derivatized product (A), the MALDI-TOF / TOF MS / MS mass spectrum of the fructose-derivatized product (B), and the working curve showing the relationship between the intensity ratio of the response difference product ions (mass-to-charge ratio of 454.2 and 484.2) and the concentrations of glucose and fructose (C).

[0041] Figure 5 The MALDI-TOF / TOF MS / MS mass spectra under different concentration ratios of glucose and fructose derivatization products.

[0042] Figure 6 These are the MALDI-TOF MS spectra of glucose / fructose in the honey (A) of Example 1 and the beverage (B) of Example 2. Specific implementation method:

[0043] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0044] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents herein are conventional commercial products in the art.

[0045] Derivatization reagent: 4-trimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-(3-aminophenylboronic acid) (TMNTA) is synthesized using cyanuric chloride as the raw material molecule and the product is obtained through a three-step substitution reaction. The reaction equation is as follows:

[0046]

[0047] The following steps are involved:

[0048] (1) Synthesis of Compound 1: 8-10 g of cyanuric chloride and 7-9 g of 1-methoxynaphthalene were added to a round-bottom flask containing 100-200 mL of toluene (0°C), followed by adding 7-9 g of aluminum chloride in batches. The mixture was stirred and reacted at room temperature for 16 h. After the reaction was completed, the reaction was quenched with 100-200 mL of 1 mol / L hydrochloric acid, and then extracted three times with dichloromethane (100-200 mL each time). The organic phases were combined, washed with saturated brine (100-200 mL), dried in vacuo, and recrystallized with petroleum ether and ethyl acetate to obtain Compound 1.

[0049] (2) Synthesis of Compound 2: 3-4 g of Compound 1 and 1-2 g of 3-aminophenylboronic acid were added to a round-bottom flask containing 20-30 mL of acetone, and 3-5 mL of 2.5 mol / L aqueous sodium hydroxide solution was added. The mixture was stirred at 0°C for 3 h. After the reaction, the reaction solution was poured into 50-100 mL of ice water, filtered, and vacuum-dried to obtain Compound 2.

[0050] (3) Synthesis of Compound 3 (TMNTA): 1-2 g of Compound 2 and 10-20 mL of 1 mol / L trimethylamine were added to a round-bottom flask containing 20-30 mL of tetrahydrofuran. The mixture was stirred at room temperature for 1 h. The reaction solution was then concentrated under vacuum, and the resulting solid was dissolved in 10-20 mL of tetrahydrofuran and stirred at room temperature for 0.5 h. The solid was filtered and washed with 10-20 mL of cold tetrahydrofuran. Purification was performed using a preparative liquid chromatography column to obtain Compound 3 (TMNTA).

[0051] The following preferred embodiment is a method for preparing compound 3 (TMNTA), which specifically comprises the following steps:

[0052] (1) Synthesis of Compound 1: 9.22 g of cyanuric chloride and 7.91 g of 1-methoxynaphthalene were added to a round-bottom flask containing 120 mL of toluene (0°C), followed by the addition of 7.99 g of aluminum chloride in portions. The mixture was stirred at room temperature for 16 h. After the reaction was completed, the reaction was quenched with 100 mL of 1 mol / L hydrochloric acid, and then extracted three times with dichloromethane (100 mL each). The organic phases were combined, washed with saturated brine (100 mL), dried in vacuo, and recrystallized from petroleum ether and ethyl acetate to obtain Compound 1.

[0053] (2) Synthesis of Compound 2: 3.06 g of Compound 1 and 1.37 g of 3-aminophenylboronic acid were added to a round-bottom flask containing 25 mL of acetone, and 4 mL of 2.5 mol / L aqueous sodium hydroxide solution was added. The mixture was stirred at 0°C for 3 h. After the reaction, the reaction solution was poured into 50 mL of ice water, filtered, and dried in vacuo to obtain Compound 2.

[0054] (3) Synthesis of Compound 3 (TMNTA): 1.25 g of Compound 2 and 15 mL of 1 mol / L trimethylamine were added to a round-bottom flask containing 20 mL of tetrahydrofuran, and the mixture was stirred at room temperature for 1 h. The reaction solution was then concentrated under vacuum, and the resulting solid was dissolved in 15 mL of tetrahydrofuran and stirred at room temperature for 0.5 h, filtered, and washed with 10 mL of cold tetrahydrofuran. Purification was performed using a liquid phase preparative column to obtain Compound 3 (TMNTA). 1 HNMR spectra see Figure 1 .

[0055] Example 1 Detection of glucose and fructose in honey

[0056] Weigh 0.1 g of honey sample, add 0.9 mL of water and vortex to dissolve, then dilute to 25,000 times with acetonitrile / water (volume ratio 1:1). The diluted sample solution contains 30 μg / mL of stable isotope internal standard [ 13 C6] Glucose. Take 10 μL of the diluted sample solution and 10 μL of TMNTA solution (TMNTA dissolved in acetonitrile at a concentration of 2 mg / mL) and vortex mix thoroughly. Then take 1 μL of the reaction solution and spot it on the MALDI target plate. After air drying at room temperature, send it to the MALDI mass spectrometer for analysis. MALDI mass spectrometry analysis uses a Smartbeam II (Nd:YAG) laser with a wavelength of 355 nm. When performing the primary mass spectrometry MALDI-TOF MS analysis, the acceleration voltage used is 19 kV, the detection mode is positive ion reflectron mode, the detection frequency is 1000 Hz, the laser energy is 40%, and each mass spectrum is the result of three superpositions of 1000 laser scans. For the secondary mass spectrometry MALDI-TOF / TOF During MS / MS analysis, the LIFT (laser-induced dissociation) mode was used, with an initial acceleration voltage of 6 kV, a LIFT cell acceleration voltage of 19 kV, a detection frequency of 200 Hz, a parent ion detection laser energy of 43%, and a daughter ion detection laser energy of 60%. Each mass spectrum is the result of three superpositions of 500 laser scans. The UV absorption measurement results of TMNTA and the commonly used MALDI small molecule matrices α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid are shown in Figure 2At a wavelength of 355 nm, the ultraviolet absorption of TMNTA is stronger than that of 2,5-dihydroxybenzoic acid and is equivalent to that of α-cyano-4-hydroxycinnamic acid. The MALDI laser wavelength used in the present invention is 355 nm, indicating that TMNTA has the properties of being a MALDI matrix.

[0057] To achieve quantitative analysis of glucose and fructose in honey, standard working curves for glucose and fructose and a working curve for the relationship between glucose and fructose concentrations were first established, respectively. Then, the "reactive matrix" TMNTA was derivatized with glucose and fructose samples to be tested, which had been spiked with stable isotope internal standards. The derivatized products of the test samples were then analyzed by MALDI-TOF MS and MALDI-TOF / TOF MS / MS. The ratio of the two response difference product ions of glucose and fructose was substituted into the obtained working curve for the relationship between glucose and fructose concentrations to calculate the concentration ratio of glucose and fructose. The mass spectral peak intensity ratio of the derivatized product of the test sample and the derivatized product of the stable isotope internal standard, as well as the concentration ratio of glucose and fructose, were substituted into the obtained glucose and fructose adduct standard working curve to calculate the concentrations of glucose and fructose in the test sample.

[0058] The establishment of standard working curves for glucose and fructose includes the following steps: preparing glucose or fructose standard solutions of different concentrations containing a fixed concentration of a stable isotope internal standard (glucose concentration is 2, 5, 10, 20, 50, 80, 100 μg / mL, and the stable isotope internal standard concentration is 30 μg / mL; fructose concentration is 5, 10, 20, 50, 80, 100 μg / mL, and the stable isotope internal standard concentration is 30 μg / mL), and performing a derivatization reaction with a "reactive matrix" TMNTA. The resulting reaction solution containing the standard derivatization product is subjected to MALDI-TOF MS analysis, and standard working curves for glucose and fructose are established, respectively, with the concentration of the glucose or fructose standard solution as the horizontal axis and the mass spectrum peak intensity ratio of the standard derivatization product to the stable isotope internal standard derivatization product as the vertical axis.

[0059] The establishment of a working curve for the relationship between glucose and fructose concentrations includes the following steps: preparing a 100 μg / mL glucose or fructose standard solution, and performing a derivatization reaction with a "reactive matrix" TMNTA, respectively; mixing the resulting reaction solutions containing glucose derivatization products and fructose derivatization products in different proportions, and performing MALDI-TOF / TOFMS / MS analysis, with the ratio of glucose and fructose concentrations as the horizontal axis and the intensity ratio of two response difference product ions (mass-to-charge ratios of 454.2 and 484.2, respectively) as the vertical axis, to obtain a working curve for the relationship between glucose and fructose concentrations, y = 0.9257x 0.3891 (See Figure 4 and Figure 5).

[0060] like Figure 6 As shown in the figure, glucose / fructose were detected in the honey sample using this method, and the spectral background was clean and free of interfering peaks.

[0061] Table 1 shows the linear range, linearity and detection limit of glucose and fructose measured by the MALDI mass spectrometry analysis method provided by the present invention, wherein each sample was measured in triplicate.

[0062] The glucose concentrations in the standard working curve were 2, 5, 10, 20, 50, 80, and 100 μg / mL, and the stable isotope internal standard concentration was 30 μg / mL; the fructose concentrations in the standard working curve were 5, 10, 20, 50, 80, and 100 μg / mL, and the stable isotope internal standard concentration was 30 μg / mL.

[0063] Table 1

[0064]

[0065] Table 2 shows the recoveries and relative standard deviations of glucose and fructose in honey measured using the MALDI mass spectrometry method provided by the present invention, wherein each sample was measured in parallel three times.

[0066] Table 2

[0067]

[0068] It can be seen that the linearity, accuracy and detection sensitivity of the quantitative detection method of the present invention are relatively good and can meet general detection requirements.

[0069] Comparative Example 1

[0070] High-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) was used to determine glucose and fructose in honey. Chromatographic conditions were: a Medium Spectrum Red Honey column, a mobile phase consisting of acetonitrile and water (77:23 by volume), a flow rate of 1.0 mL / min, a column temperature of 30°C, and an injection volume of 5 μL. The ELSD conditions were: a drift tube temperature of 85°C, high-purity nitrogen as the carrier gas, a carrier gas flow rate of 2.50 SLM, and a gain of 1.0.

[0071] Table 3 is a comparison of the glucose and fructose concentrations in honey measured using the MALDI mass spectrometry method (MALDI-MS) provided in Example 1 and the high performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD) of Comparative Example 1.

[0072] Table 3

[0073]

[0074]

[0075] As can be seen, the glucose and fructose concentrations determined by the present invention are essentially the same as those determined by HPLC-ELSD. However, unlike the time-consuming chromatographic separation required by HPLC-ELSD, the present invention does not require this chromatographic separation, and detection can be performed immediately after mixing the sample solution with the reactive matrix solution. This method offers the advantages of simple operation, rapidity, and high throughput. Therefore, the present invention has broad application value for the quantitative detection of glucose and fructose.

[0076] Example 2 Detection of glucose and fructose in beverages

[0077] 0.1 mL of beverage sample was measured and diluted to 5000 times with acetonitrile / water (volume ratio 1:1). The diluted sample solution contained 30 μg / mL of stable isotope internal standard [ 13 C6] Glucose. Take 10 μL of the diluted sample solution and 10 μL of TMNTA solution (TMNTA dissolved in acetonitrile at a concentration of 2 mg / mL) and vortex mix thoroughly. Then take 1 μL of the reaction solution and spot it on the MALDI target plate. After air drying at room temperature, send it to the MALDI mass spectrometer for analysis. MALDI mass spectrometry analysis uses a Smartbeam II (Nd:YAG) laser with a wavelength of 355 nm. When performing the primary mass spectrometry MALDI-TOF MS analysis, the acceleration voltage used is 19 kV, the detection mode is positive ion reflection mode, the detection frequency is 1000 Hz, the laser energy is 40%, and each mass spectrum is the result of three superpositions of 1000 laser scans. For the secondary mass spectrometry MALDI-TOF / TOF During MS / MS analysis, the LIFT (laser-induced dissociation) mode was used, with an initial acceleration voltage of 6 kV, a LIFT cell acceleration voltage of 19 kV, a detection frequency of 200 Hz, a parent ion detection laser energy of 43%, and a daughter ion detection laser energy of 60%. Each mass spectrum was the result of three superpositions of 500 laser scans. Figure 6 As shown in the figure, glucose and fructose were detected in the beverage sample using this method, and the spectral background was clean and free of interference.

[0078] Example 3

[0079] The method is the same as Example 1, except that: the MALDI mass spectrometry detection conditions are as follows: a Nd:YAG laser with a wavelength of 266 nm is used, the primary mass spectrometer (MALDI-TOF MS) uses an accelerating voltage of 19 kV, the detection mode is a positive ion reflectron mode, the detection frequency is 800 Hz, and the laser energy is 30%; the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) uses an initial accelerating voltage of 5 kV, a dissociation cell accelerating voltage of 18 kV, a detection frequency of 200 Hz, a parent ion detection laser energy of 30%, and a daughter ion detection laser energy of 50%.

[0080] Example 4

[0081] The method is the same as Example 1, except that: the MALDI mass spectrometry detection conditions are as follows: a nitrogen molecule laser with a wavelength of 337 nm is used, the primary mass spectrometer (MALDI-TOF MS) uses an accelerating voltage of 21 kV, the detection mode is a positive ion reflectron mode, the detection frequency is 1200 Hz, and the laser energy is 50%; the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) uses an initial accelerating voltage of 8 kV, a dissociation cell accelerating voltage of 20 kV, a detection frequency of 500 Hz, a parent ion detection laser energy of 50%, and a daughter ion detection laser energy of 70%.

[0082] The description of the above embodiments is only used to help understand the technical solution and core ideas of the present invention. It should be pointed out that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A compound, characterized in that The structural formula of the compound is shown in Formula 1: Formula 1.

2. The method for preparing the compound according to claim 1, characterized in that Using cyanuric chloride as a raw material molecule, a three-step substitution reaction is performed to obtain compound 3, which is the compound described. The chemical reaction equation is shown in Formula 2: Formula 2.

3. The preparation method according to claim 2, comprising the steps of: (1) Synthesis of Compound 1: Cyanuric chloride and 1-methoxynaphthalene were added to a reaction vessel containing toluene, wherein the molar ratio of cyanuric chloride to 1-methoxynaphthalene was 0.5-2.0:1, and the mass volume ratio of cyanuric chloride to toluene was 4-10:100 g / mL. Aluminum chloride was then added in portions, wherein the molar ratio of 1-methoxynaphthalene to aluminum chloride was 0.5-2.0:

1. The mixture was stirred at room temperature for 12-20 h. After the reaction was completed, the reaction was quenched with hydrochloric acid, and the organic phases were extracted and combined, washed, dried under vacuum, and then recrystallized to obtain Compound 1. (2) Synthesis of Compound 2: Compound 1 and 3-aminophenylboronic acid were added to a reaction vessel containing acetone, wherein the molar ratio of Compound 1 to 3-aminophenylboronic acid was 0.5-2.0:1, and the mass volume ratio of 3-aminophenylboronic acid to acetone was 1:10-30 g / mL. A sodium hydroxide aqueous solution was added, wherein the molar ratio of 3-aminophenylboronic acid to sodium hydroxide was 0.5-2.0:

1. The mixture was stirred at 0°C for 2-4 h. After the reaction was completed, the reaction solution was poured into ice water, filtered, and vacuum dried to obtain Compound 2. (3) Synthesis of compound 3: Compound 2 and trimethylamine were added to a reaction vessel containing tetrahydrofuran, with the mass volume ratio of compound 2 to tetrahydrofuran being 1:10-30 g / mL and the concentration of trimethylamine in the reaction system being 1 / 5-2 / 3 mol / L. The mixture was stirred at room temperature for 0.5-1.5 h. The reaction solution was then concentrated in vacuo, and the resulting solid was dissolved in tetrahydrofuran and stirred at room temperature for 0.25-0.75 h. The solid was filtered and washed with cold tetrahydrofuran, and purified to obtain compound 3.

4. Use of the compound according to claim 1 in the quantitative detection of glucose and fructose in food.

5. A rapid, high-throughput method for quantitative detection of glucose and fructose in food, characterized in that: The method comprises the following steps: using the compound according to claim 1 as a reactive matrix to undergo a derivatization reaction with glucose and fructose, and detecting the derivatized products by MALDI-TOF MS and MALDI-TOF / TOF MS / MS to perform qualitative and quantitative analysis of the target.

6. The quantitative detection method according to claim 5, characterized in that The following steps are involved: The compound was dissolved in acetonitrile solution and then vortexed with glucose and fructose for derivatization reaction; After the derivatization reaction, the reaction solution was spotted on a target plate, air-dried at room temperature, and then sent to a MALDI mass spectrometer for quantitative detection of glucose and fructose using the stable isotope internal standard method. The relationship between glucose and fructose content was analyzed using MALDI-TOF / TOF MS / MS.

7. The quantitative detection method according to claim 6, characterized in that The MALDI mass spectrometry detection conditions are as follows: a laser with a wavelength of 250-360 nm is used, the acceleration voltage used in the first-stage mass spectrometry is 19-21 kV, the detection mode is positive ion reflectron mode, the detection frequency is 800-1200 Hz, and the laser energy is 30%-50%; the initial acceleration voltage used in the second-stage mass spectrometry is 5-8 kV, the dissociation cell acceleration voltage is 18-20 kV, the detection frequency is 200-500 Hz, the parent ion detection laser energy is set to 30%-50%, and the daughter ion detection laser energy is set to 50%-70%.

8. The quantitative detection method according to claim 6, characterized in that When MALDI mass spectrometry is used to quantitatively detect glucose and fructose in actual samples, [ 13 C6] glucose was used as a stable isotope internal standard.

9. The quantitative detection method according to claim 6, characterized in that The specific steps include: S1. Glucose or fructose standard solutions of different concentrations containing a fixed concentration of a stable isotope internal standard are prepared, and derivatized with the compound. The resulting reaction solution containing the standard derivatized product is subjected to MALDI-TOF MS analysis, with the concentration of the glucose or fructose standard solution as the abscissa and the intensity ratio of the mass spectral peak of the standard derivatized product to the stable isotope internal standard derivatized product as the ordinate, to establish standard working curves for glucose and fructose, respectively. The standard working curves for glucose and fructose are added together to obtain a standard working curve containing both glucose and fructose concentrations. S2. Glucose and fructose are prepared at the same concentrations, and derivatized with the compounds. The resulting reaction solutions containing glucose-derivative products and fructose-derivative products are mixed in different proportions and analyzed by MALDI-TOF / TOF MS / MS. A working curve for the relationship between glucose and fructose concentrations is established, with the ratio of glucose to fructose concentrations as the abscissa and the intensity ratio of the two differentially responsive product ions as the ordinate. S3. Derivatizing the compound with glucose and fructose test samples to which stable isotope internal standards are added, and then subjecting the derivatized products of the test samples to MALDI-TOF MS and MALDI-TOF / TOF MS / MS analysis. Substituting the ratio of the two response difference daughter ions of glucose and fructose into the working curve obtained in step S2, thereby calculating the concentration ratio of glucose and fructose; substituting the mass spectrum peak intensity ratio of the derivatized product of the test sample and the derivatized product of the stable isotope internal standard and the concentration ratio of glucose and fructose into the standard working curve obtained in step S1, thereby calculating the concentrations of glucose and fructose in the test sample.

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