Rapid and high-flux quantitative detection method for glucose and fructose in food

Abstract

The invention discloses a rapid and high-throughput quantitative detection method for glucose and fructose in food. A rapid and high-flux quantitative detection method for glucose and fructose in food comprises the following steps: carrying out derivatization reaction on a compound TMNTA as a reactive matrix and glucose and fructose, and detecting derivatization products through MALDI-TOF MS and MALDI-TOF / TOF MS / MS so as to carry out qualitative and quantitative analysis on a target object. The quantitative detection method provided by the invention is simple, convenient and rapid to operate, and the whole 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 a food sample can be met.

Description

Technical field:

[0001] The 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 exist naturally or are added as sweeteners in a variety of foods. They are one of the important nutrients in food and provide energy for human life activities. The content of glucose and fructose is an important quality indicator of some foods. For example, the "National Food Safety Standard Honey" (GB14963-2011) stipulates that the sum of the glucose and fructose content in honey should be ≥60% (g / 100g), and the "Orange Juice and Orange Juice Beverage" (BG / T21731-2008) stipulates that the glucose and fructose content should be within the range of 20.0-35.0g / kg and the ratio of glucose to fructose content should be ≤1.0. Therefore, accurate monitoring of the content of glucose and fructose in food is of great significance to food quality evaluation and food quality control.

[0003] Since glucose and fructose lack good UV absorption groups in their structures, it is particularly difficult to detect them directly with common UV detectors. Therefore, the more common detection methods currently include high performance liquid chromatography (HPLC) combined with special detectors such as differential refractive index detector (RID), evaporative light scattering detector (ELSD), charged aerosol detector (CAD), ion chromatography, liquid chromatography-mass spectrometry, etc. These methods all require time-consuming chromatographic separation processes and cannot achieve rapid high-throughput detection. Matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS) has the advantages of accuracy, rapid analysis, high sensitivity, high throughput, simple sample preparation, etc., and is expected to play an important role in the rapid and high-throughput detection of glucose and fructose in food. However, this technology has problems such as matrix signal interference in the low mass-to-charge ratio region, poor reproducibility of mass spectrometry signals, and difficulty in quantifying isomers separately, which poses great difficulties for the quantitative detection of glucose and fructose in food. There is 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 faced by traditional food testing technology. Therefore, it is urgent to establish an accurate, simple, fast, high-throughput, and highly sensitive efficient analytical method for 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 process and low detection throughput in the prior art, and provides a rapid and high-throughput quantitative detection method for glucose and fructose in food. The quantitative detection method proposed by the present invention is simple and rapid to operate, and the entire experimental process can be completed within 2 minutes; the quantitative result is accurate, and can meet the 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, the compound is 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)), and the structural formula is 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, and its 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 are added to a reaction vessel containing toluene, wherein the molar ratio of cyanuric chloride to 1-methoxynaphthalene is 0.5-2.0:1, and the mass volume ratio of cyanuric chloride to toluene is 4-10:100 g / mL. Subsequently, aluminum chloride is added in portions, wherein the molar ratio of 1-methoxynaphthalene to aluminum chloride is 0.5-2.0:1. The mixture is stirred at room temperature for reaction for 12-20 hours. After the reaction is completed, the reaction is quenched with hydrochloric acid, and the organic phases are extracted and combined, washed, vacuum dried, and then recrystallized to obtain Compound 1;

[0012] (2) Synthesis of Compound 2: Compound 1 and 3-aminophenylboronic acid are added to a reaction vessel containing acetone, wherein the molar ratio of Compound 1 to 3-aminophenylboronic acid is 0.5-2.0:1, and the mass volume ratio of 3-aminophenylboronic acid to acetone is 1:10-30 g / mL. A sodium hydroxide aqueous solution is added, wherein the molar ratio of 3-aminophenylboronic acid to sodium hydroxide is 0.5-2.0:1. The mixture is stirred at 0° C. for reaction for 2-4 h. After the reaction is completed, the reaction solution is 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 and washed with cold tetrahydrofuran, and purified to obtain Compound 3.

[0014] Further 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, the molar ratio of cyanuric chloride to 1-methoxynaphthalene being 1:1, and the mass volume ratio of cyanuric chloride to toluene being 7-8:100 g / mL, and then aluminum chloride was added in portions, the molar ratio of 1-methoxynaphthalene to aluminum chloride being 5:6, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction was quenched with hydrochloric acid, and the organic phases were extracted and combined, washed, vacuum dried, 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 at 0°C for 3 h. 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 and 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] The fourth object of the present invention is to provide a rapid, high-throughput method for quantitatively detecting glucose and fructose in food, comprising the following steps: 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.

[0020] The present invention adopts a boronic acid-modified compound as a reactive matrix to react with glucose and fructose for a specific derivatization reaction, so that the signal response of the target object after derivatization is enhanced and the detection sensitivity is improved; no additional matrix needs to be added after derivatization, thereby solving the problem of matrix background interference; a stable isotope compound is used as an internal standard, so that the fluctuation of the mass spectrometry signal and the matrix effect of the sample can be effectively corrected, thereby realizing accurate quantitative analysis; and 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 realizing the separate quantitative analysis of glucose and fructose, thereby solving the problem of quantitative detection of isomers by MALDI mass spectrometry.

[0021] The reaction formula of 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, and then vortex mixing with glucose and fructose to perform a (specific) derivatization reaction; after the derivatization reaction, taking the reaction solution and spotting it on a target plate, air-drying it at room temperature, and then sending it into a MALDI mass spectrometer, using a stable isotope internal standard method to perform MALDI mass spectrometry quantitative detection of glucose and fructose; and using MALDI-TOF / TOF MS / MS to analyze the relationship between the glucose and fructose contents.

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

[0025] Further preferably, the MALDI mass spectrometry detection conditions are: using a laser with a wavelength of 266-337nm, the acceleration voltage used by the primary mass spectrometer (MALDI-TOF MS) is 19-21kV, the detection mode is the positive ion reflection mode, the detection frequency is 800-1200Hz, and the laser energy is 30%-50%; the initial acceleration voltage used by the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) is 5-8kV, the dissociation cell acceleration voltage is 18-20kV, the detection frequency is 200-500Hz, the parent ion detection laser energy is set to 30%-50%, and the daughter ion detection laser energy 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; and each mass spectrum detected by the secondary mass spectrometry (MALDI-TOF / TOF MS / MS) is the average result of 300-1000 laser scans.

[0027] In order to achieve accurate quantitative analysis, stable isotope internal standard method was used to perform MALDI mass spectrometry to quantitatively detect glucose and fructose. Secondary mass spectrometry (MALDI-TOF / TOF MS / MS) was used to analyze the relationship between glucose and fructose content.

[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 targets and the matrix effects caused by the samples to provide accurate quantitative data.

[0029] Preferably, the quantitative detection method is characterized by comprising the following steps:

[0030] S1. Prepare glucose or fructose standard solutions of different concentrations containing a fixed concentration of a stable isotope internal standard, and perform a derivatization reaction with the compound, and perform MALDI-TOF MS analysis on the obtained reaction solution containing the standard derivatization product, and establish standard working curves of glucose and fructose respectively with the concentration of the glucose or fructose standard solution as the horizontal coordinate and the intensity ratio of the mass spectrum peak of the standard derivatization product to the stable isotope internal standard derivatization product as the vertical coordinate, and add the standard working curves of glucose and fructose to obtain a standard working curve containing both glucose and fructose concentrations;

[0031] S2. Glucose and fructose of the same concentration are prepared respectively, and the compounds are used for derivatization reaction respectively. The obtained reaction solution containing glucose derivatization product and the solution containing fructose derivatization product are mixed in different proportions and then subjected to MALDI-TOF / TOF MS / MS analysis. The ratio of glucose and fructose concentrations is used as the horizontal axis, and the intensity ratio of the two response difference daughter ions is used as the vertical axis to establish a working curve of the relationship between glucose and fructose concentrations;

[0032] S3, subjecting the compound to a derivatization reaction with glucose and fructose test samples to which stable isotope internal standards are added, and then subjecting the derivatization 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 derivatization product of the test sample and the derivatization 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 two daughter ions of glucose and fructose derivatives with different responses to obtain the concentration relationship between 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 chromatographic separation process.

[0036] 3. The quantitative detection method proposed in the present invention is quick and easy to operate. The sample to be tested is derivatized with the "reactive matrix" TMNTA and then 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 MALDI-TOF / TOF MS / MS mass spectrum of glucose derivatization product (A), MALDI-TOF / TOF MS / MS mass spectrum of fructose derivatization product (B), working curve of the relationship between the intensity ratio of response difference product ions (mass-to-charge ratio of 454.2 and 484.2) and the concentration of glucose and fructose (C).

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

[0042] Figure 6 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, rather than to limit the present invention.

[0044] Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments 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: The synthesis of 4-trimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-(3-aminophenylboronic acid) (TMNTA) uses cyanuric chloride as a raw material molecule and obtains the product 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, and the mixture was stirred 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 with dichloromethane three times (100-200 mL each time), the organic phases were combined, washed with saturated brine (100-200 mL), vacuum dried, 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 sodium hydroxide aqueous solution was added, and 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, and the mixture was stirred at room temperature for 1 h. The reaction solution was then concentrated in vacuo, and the resulting solid was dissolved in 10-20 mL of tetrahydrofuran and stirred at room temperature for 0.5 h, filtered, and washed with 10-20 mL of cold tetrahydrofuran. Purification was performed using a liquid phase preparative 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 adding 7.99 g of aluminum chloride in portions, and 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 time), the organic phases were combined, washed with saturated brine (100 mL), vacuum dried, and recrystallized with 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 sodium hydroxide aqueous solution was added, and 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 vacuum dried 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 in vacuo, 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), which 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, the concentration is 2 mg / mL) and vortex mix, then take 1 μL of the reaction solution and spot it on the MALDI target plate, air-dry it at room temperature and 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 the 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; perform the secondary mass spectrometry MALDI-TOF / TOF In MS / MS analysis, the LIFT (laser induced dissociation) mode was used, the initial acceleration voltage was 6 kV, the LIFT cell acceleration voltage was 19 kV, the detection frequency was 200 Hz, the parent ion detection laser energy was set to 43%, the daughter ion detection laser energy was set to 60%, and each mass spectrum was 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 property of being a MALDI matrix.

[0057] In order to achieve the quantitative analysis of glucose and fructose in honey, firstly, the standard working curves of glucose and fructose and the working curve of the relationship between glucose and fructose concentrations were established respectively, then the "reactive matrix" TMNTA was subjected to derivatization reaction with the glucose and fructose test samples added with stable isotope internal standards, and then the derivatization products of the test samples were subjected to MALDI-TOF MS and MALDI-TOF / TOF MS / MS analysis, and the ratio of the two response difference daughter ions of glucose and fructose was substituted into the obtained working curve of the relationship between glucose and fructose concentrations, so as to calculate the concentration ratio of glucose and fructose; the mass spectrum peak intensity ratio of the derivatization product of the test sample and the derivatization product of the stable isotope internal standard and the concentration ratio of glucose and fructose were substituted into the obtained glucose and fructose addition standard working curve, so as to calculate the concentrations of glucose and fructose in the test sample.

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

[0059] The establishment of the working curve of the relationship between glucose and fructose concentrations includes the following steps: preparing a glucose or fructose standard solution with a concentration of 100 μg / mL, and performing a derivatization reaction with a "reactive matrix" TMNTA, respectively; mixing the resulting reaction solution containing glucose derivatization products and the fructose derivatization reaction product solution in different proportions, and then performing MALDI-TOF / TOFMS / MS analysis; taking the ratio of glucose and fructose concentrations as the horizontal coordinate, and the intensity ratio of two response difference daughter ions (mass-to-charge ratios of 454.2 and 484.2, respectively) as the vertical coordinate, to obtain a working curve of 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 was detected in the honey sample using this method, and the spectrum 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 three times in parallel.

[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 spiked recoveries and relative standard deviations of glucose and fructose in honey measured by the MALDI mass spectrometry analysis method provided by the present invention, wherein each sample was measured in parallel 3 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 detect glucose and fructose in honey. The chromatographic conditions were as follows: the chromatographic column was a medium spectrum red honey column, the mobile phase was acetonitrile and water (volume ratio was 77:23), the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the injection volume was 5 μL. The conditions of the evaporative light scattering detector were as follows: the drift tube temperature was 85 °C, the carrier gas was high purity nitrogen, the carrier gas flow rate was 2.50 SLM, and the gain value was 1.0.

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

[0072] Table 3

[0073]

[0074]

[0075] It can be seen that the concentrations of glucose and fructose determined by the present invention and the HPLC-ELSD method are basically consistent; however, compared with the time-consuming chromatographic separation process required by the HPLC-ELSD method, the present invention does not require a chromatographic separation process, and the sample solution can be detected after being mixed with the "reactive" matrix solution. The present invention has the advantages of simple operation, rapidity, and high throughput. Therefore, the present invention has a wide range of 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, concentration is 2 mg / mL) and vortex mix, then take 1 μL of the reaction solution and spot it on the MALDI target plate, air dry at room temperature and 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 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; perform 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 samples using this method, and the spectrum 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: a Nd:YAG laser with a wavelength of 266 nm is used, the acceleration voltage used in the primary mass spectrometer (MALDI-TOF MS) is 19 kV, the detection mode is the positive ion reflection mode, the detection frequency is 800 Hz, and the laser energy is 30%; the initial acceleration voltage used in the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) is 5 kV, the dissociation cell acceleration voltage is 18 kV, the detection frequency is 200 Hz, the parent ion detection laser energy is set to 30%, and the daughter ion detection laser energy is set to 50%.

[0080] Example 4

[0081] The method is the same as Example 1, except that: the MALDI mass spectrometry detection conditions are: a nitrogen molecule laser with a wavelength of 337 nm is used, the acceleration voltage used in the primary mass spectrometer (MALDI-TOF MS) is 21 kV, the detection mode is the positive ion reflection mode, the detection frequency is 1200 Hz, and the laser energy is 50%; the initial acceleration voltage used in the secondary mass spectrometer (MALDI-TOF / TOF MS / MS) is 8 kV, the dissociation cell acceleration voltage is 20 kV, the detection frequency is 500 Hz, the parent ion detection laser energy is set to 50%, and the daughter ion detection laser energy is set to 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 technicians in this technical field, 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 compound is named 4-trimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-(3-aminophenylboronic acid), and its structural formula is shown in 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 above. The chemical reaction equation is shown in Formula 2:

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

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

1. The mixture is stirred at 0° C. for reaction for 2-4 h. After the reaction is completed, the reaction solution is 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, 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 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 described in claim 1 as a reactive matrix to undergo a derivatization reaction with glucose and fructose, and detecting the derivatized product 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 compounds were dissolved in acetonitrile solution and then vortexed with glucose and fructose for derivatization reaction; After the derivatization reaction, the reaction solution was spotted on the target plate, air-dried at room temperature, and then sent to the MALDI mass spectrometer. The stable isotope internal standard method was used to perform MALDI mass spectrometry quantitative detection of glucose and fructose; 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 250-360nm is used, the acceleration voltage used by the primary mass spectrometry is 19-21kV, the detection mode is the positive ion reflection mode, the detection frequency is 800-1200Hz, and the laser energy is 30%-50%; the initial acceleration voltage used by the secondary mass spectrometry is 5-8kV, the dissociation cell acceleration voltage is 18-20kV, the detection frequency is 200-500Hz, 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. Prepare glucose or fructose standard solutions of different concentrations containing a fixed concentration of a stable isotope internal standard, and perform a derivatization reaction with the compound, and perform MALDI-TOF MS analysis on the obtained reaction solution containing the standard derivatization product, and establish standard working curves of glucose and fructose respectively with the concentration of the glucose or fructose standard solution as the horizontal coordinate and the intensity ratio of the mass spectrum peak of the standard derivatization product to the stable isotope internal standard derivatization product as the vertical coordinate, and add the standard working curves of glucose and fructose to obtain a standard working curve containing both glucose and fructose concentrations; S2. Glucose and fructose of the same concentration are prepared respectively, and the compounds are used for derivatization reaction respectively. The obtained reaction solution containing glucose derivatization product and the solution containing fructose derivatization product are mixed in different proportions and then subjected to MALDI-TOF / TOF MS / MS analysis. The ratio of glucose and fructose concentrations is used as the horizontal axis, and the intensity ratio of the two response difference daughter ions is used as the vertical axis to establish a working curve of the relationship between glucose and fructose concentrations; S3, subjecting the compound to a derivatization reaction with glucose and fructose test samples to which stable isotope internal standards are added, and then subjecting the derivatization 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 derivatization product of the test sample and the derivatization 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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