2-aminoisophthalic acid as a maldi matrix for the detection of oligosaccharide additives in food

Abstract

The application discloses application of 2-amino isophthalic acid (2-AIA) as a MALDI matrix in characterization of oligosaccharide additives in food. The skeleton structure of 2-AIA has a strong ultraviolet absorption group and a protonation site, which is beneficial to laser energy transmission and desorption ionization process of the measured object. Meanwhile, 2-AIA has multiple polar functional groups, which can effectively enhance the directional combination of the hydroxyl group of 2-AIA and oligosaccharide, and improve the anti-interference ability of impurities in a complex system. In addition, 2-AIA and a traditional organic matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) are combined as a binary matrix (CHCA / 2-AIA), which can play a synergistic role of the double matrix, significantly improve the detection sensitivity of oligosaccharide. On the other hand, the combination of CHCA and 2-AIA with different pH values can significantly improve the uniformity of the co-crystallization, and improve the accuracy and reproducibility of quantitative detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food additive analysis using MALDI (Matrix Assisted Laser Desorption Ionization) mass spectrometry technology, and in particular to the application of 2-aminoisophthalic acid (2-AIA) as a MALDI matrix for detecting oligosaccharide additives in food. BACKGROUND

[0002] With the rapid development of the food industry, oligosaccharides are widely added to dairy products, infant formula, functional beverages and baked products due to their functional properties such as prebiotics, dietary fiber fortification and low-calorie sweetness. However, the physicochemical properties (such as the degree of polymerization distribution) of oligosaccharide additives in commercially available foods are closely related to factors such as their type, source, extraction method and enzymatic conditions. Therefore, it is necessary to control the quality of foods containing oligosaccharide additives in order to ensure food safety.

[0003] Current characterization techniques for oligosaccharides in foods mainly rely on high-performance liquid chromatography, capillary electrophoresis and traditional mass spectrometry. However, these methods generally have the following bottlenecks: (1) the pretreatment is complicated and can easily lead to the loss of trace oligosaccharides; (2) the sensitivity is insufficient, which cannot meet the detection limit requirements for trace additives; (3) the analysis is time-consuming, which cannot meet the high-throughput requirements of industrial production.

[0004] Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has unique advantages such as high throughput, high sensitivity and no need for complex pretreatment, which has opened up new ways for the complete analysis of macromolecular compounds (molecular weight > 1000) in complex systems. However, the ionization efficiency of oligosaccharides is poor, and the use of traditional MALDI matrices (such as 2,5-dihydroxybenzoic acid) for analysis has defects such as uneven co-crystallization, poor reproducibility and low detection sensitivity. Although researchers have tried to improve these problems by developing new matrices, existing solutions still have limitations. For example, although reactive matrices can improve ionization efficiency, they are only suitable for reducing sugars with reducing ends as reaction groups, and are not suitable for a variety of oligosaccharides in foods.

[0005] Therefore, there is an urgent need to design and develop an oligosaccharide analysis strategy based on a new MALDI matrix, which has the advantages of high sensitivity, good universality and uniform co-crystallization, in order to meet the rapid and high-throughput detection needs of oligosaccharide additives in the food industry. SUMMARY

[0006] The present application provides the application of 2-aminoisophthalic acid as a MALDI matrix for characterizing oligosaccharide additives in food.

[0007] In this invention, 2-aminoisophthalic acid (2-AIA) is used as the MALDI matrix. This compound is an aromatic carboxylic acid compound containing amino groups. Its skeleton structure has both strong ultraviolet absorption groups and protonation sites, which is beneficial to the transfer of laser energy and the desorption and ionization process of the analyte. At the same time, 2-AIA has multiple polar functional groups, which can effectively enhance its directional binding with the hydroxyl groups of oligosaccharides and improve the resistance to impurity interference in complex systems. The traditional organic matrix α-cyano-4-hydroxycinnamic acid (CHCA) and 2-AIA are used as a binary matrix (CHCA / 2-AIA), which can significantly improve the detection sensitivity of oligosaccharides and realize rapid and high-throughput analysis of oligosaccharide additives in food. Among them, CHCA can synergistically promote ionization and broaden the detection range of oligosaccharides. In addition, the combined use of CHCA with different pH values ​​and 2-AIA can significantly improve the uniformity of co-crystallization and improve the accuracy and reproducibility of quantitative detection. The structural formula of 2-AIA in this invention is shown in formula (1):

[0008]

[0009] 2-Amino-isophthalic acid (2-AIA) was used as a MALDI matrix, and combined with α-cyano-4-hydroxycinnamic acid (CHCA) as a binary matrix for characterizing the application of oligosaccharide additives in food.

[0010] In this invention, the molecular backbone of 2-AIA is a complex structure of isophthalic acid and an amino group at the 2-position, which is beneficial for the efficient absorption of 355 nm laser energy and improves the desorption efficiency of oligosaccharides. Simultaneously, the amino and carboxylic acid groups anchor oligosaccharide molecules in the complex system through multiple hydrogen bonds, excluding interference from impurity molecules. The strong electron-withdrawing cyano group of CHCA enhances the conjugated system, improves the absorption efficiency of laser energy, and broadens the detection range of oligosaccharides. Both 2-AIA and CHCA are highly efficient matrices for MALDI mass spectrometry detection, and their different pH levels can further enhance the compactness of the co-crystallization, improving the accuracy and reproducibility of quantitative detection.

[0011] In this invention, 2-AIA is used as a MALDI matrix, and its combination with CHCA serves as a binary matrix for characterizing oligosaccharide additives in food. The specific steps are as follows:

[0012] (1) Reagent preparation

[0013] A 2-AIA solution was prepared using dimethyl sulfoxide and acetone as a mixed solvent; a CHCA / 2-AIA binary matrix solution was prepared using CHCA as a solute and 2-AIA solution as a solvent; water was added to the food samples (including fruit juice, milk and yogurt) to dilute them and obtain the corresponding sample solutions.

[0014] (2) Spotting

[0015] Equal volumes of diluted sample solution and CHCA / 2-AIA binary matrix solution were taken and spotted using the dry drop method or thin-layer method to obtain the spotted target plate.

[0016] (3) Drying

[0017] After spotting, the target plate is placed at room temperature until the mixed sample spots are completely dry.

[0018] (4) Mass spectrometry detection

[0019] The dried target plate was sent into a MALDI-TOF-MS mass spectrometer and detected using positive ion reflectance mode.

[0020] In step (1), the dimethyl sulfoxide and acetone used to dissolve the matrix are analytical grade or chromatographic grade solvents;

[0021] In the mixed solvent of dimethyl sulfoxide and acetone, the volume ratio of dimethyl sulfoxide is 12-18%, more preferably 15%;

[0022] The concentration of 2-AIA in the CHCA / 2-AIA binary matrix solution is 0.01–0.03 mol / L, more preferably 0.02 mol / L;

[0023] The molar ratio of CHCA to 2-AIA in the CHCA / 2-AIA binary matrix solution is 1.1 to 2.9:1, more preferably 2:1;

[0024] The solvent used to dilute food samples is ultrapure water or double-distilled water;

[0025] In step (2), the sample solution is spotted in a volume of 0.5 to 1.2 μL, more preferably 1 μL;

[0026] The spotting volume of the CHCA / 2-AIA binary matrix solution is 0.5–1.2 μL, more preferably 1 μL;

[0027] The dry-drop method involves sequentially spotting the binary matrix solution and the sample solution into the same spotting well on the target plate, and then using a pipette to repeatedly aspirate and tap the polished target plate 10 to 20 times to mix them.

[0028] The thin-layer method involves first spotting a binary matrix solution onto the sample well of the target plate, drying it, and then spotting the sample solution onto the upper layer of the matrix crystals.

[0029] In step (3), the room temperature is 20-25°C.

[0030] The "MALDI mass spectrometry" mentioned in this invention refers to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), abbreviated as MALDI mass spectrometry. The working principle of MALDI is to irradiate the co-crystallization formed by the sample and matrix with a laser. The matrix absorbs energy from the laser and transfers it to the sample molecules, causing them to ionize. MALDI is a soft ionization technique suitable for the determination of biomolecules.

[0031] The “binary matrix” mentioned in this invention refers to a novel matrix system formed by combining two organic MALDI matrices. This matrix system can not only effectively improve the problem of uneven crystallization of a single matrix, but also significantly enhance the ionization efficiency of carbohydrates through synergistic effects.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1,2-AIA is an amino-containing aromatic carboxylic acid compound with a skeleton structure containing both strong ultraviolet absorbing groups and protonation sites, which is beneficial for laser energy transfer and the desorption and ionization process of the analyte, making it very suitable as a MALDI matrix.

[0034] 2-AIA possesses both amino and carboxyl polar functional groups, which facilitates its directional binding with the hydroxyl groups of oligosaccharides through multiple hydrogen bonds. This enhances the desorption and ionization efficiency of oligosaccharides from complex food samples and strengthens their resistance to interference from impurities, making it highly suitable as a matrix for carbohydrate compounds.

[0035] 3. Both CHCA and 2-AIA are highly efficient matrices for MALDI mass spectrometry detection. The combination of the two can exert the synergistic effect of binary matrices and significantly improve the detection sensitivity of oligosaccharides. CHCA / 2-AIA can achieve oligosaccharide analysis at a level as low as 10 fmol, which is 50 times higher than that of the traditional carbohydrate matrix 2,5-dihydroxybenzoic acid (DHB).

[0036] 4. CHCA and 2-AIA have different pH levels. Using them together as a binary matrix can significantly improve the uniformity of co-crystallization between the matrix and the sample, thus enhancing the accuracy and stability of quantitative detection (RSD < 10%). The CHCA / 2-AIA binary matrix is ​​suitable for the qualitative and quantitative detection of oligosaccharides, exhibiting good linearity in the range of 0.625–10 pmol / μL. 2 >0.999. Attached Figure Description

[0037] Figure 1 The NMR spectrum and high-resolution ESI mass spectrum of 2-AIA; Figure 1 A is the 1H NMR spectrum of 2-AIA; Figure 1B is the 13C NMR spectrum of 2-AIA; Figure 1 C represents the high-resolution ESI mass spectrum in 2-AIA negative ion mode;

[0038] Figure 2 The UV absorption spectra of DHB and 2-AIA at the same molar concentration are shown.

[0039] Figure 3 To detect the MALDI mass spectrum of malthexaose (G6); Figure 3 A is the MALDI mass spectrum of G6 detected using DHB as the matrix; Figure 3 B is the MALDI mass spectrum of G6 detected using 2-AIA as the matrix;

[0040] Figure 4 The effect of different sample preparation conditions on the detection of G6 in 2-AIA matrix; Figure 4 A shows the MALDI mass spectra of G6 detected in different mixed solvents under positive ion reflectance mode; Figure 4 B represents the effect of the volume ratio of dimethyl sulfoxide on the detection of G6. Figure 4 The effect of C on the concentration of 2-AIA on G6 detection; Figure 4 D represents the impact of the spotting method on G6 detection; Figure 4 E represents the effect of drying temperature on G6 detection;

[0041] Figure 5 MALDI mass spectra of G6 and co-crystallization morphologies of G6 with different traditional organic matrices and 2-AIA were detected in positive ion reflectance mode.

[0042] Figure 6 The effect of different molar ratios of CHCA and 2-AIA on the detection of G6; Figure 6 A represents the impact on the G6 signal strength; Figure 6 B represents the effect on co-crystallization;

[0043] Figure 7 To determine the detection limits of different oligosaccharides using DHB and CHCA / 2-AIA as MALDI matrices, respectively; Figure 7 A and B represent the detection limits for maltoheptaose (G7); Figure 7 C and D represent the detection limits for NGA3; Figure 7 E and F are the detection limits of NA2; Figure 7 G and H are the detection limits of Man-8; where Figure 7 A, C, E, and G are MALDI spectra detected using CHCA / 2-AIA as the matrix; Figure 7 B, D, F, H are MALDI spectra detected using DHB as the matrix;

[0044] Figure 8 To assess the suitability of using CHCA / 2-AIA as the MALDI matrix for testing oligosaccharide mixtures D2000;

[0045] Figure 9 To determine the reproducibility of the signal when detecting G7 using DHB and CHCA / 2-AIA as matrices and the quantitative curve fitted after measuring NGA3, NA2 and Man-8; Figure 9 A is the normalized plot of the mean intensity and RSD value of the peak obtained from 45 random detections at the same sampling well; Figure 9 B is the quantitative curve for NGA3 detection in the CHCA / 2-AIA matrix; Figure 9 C represents the quantitative curve for NA2 detection using the CHCA / 2-AIA matrix; Figure 9 D is the quantitative curve for the detection of Man-8 by the CHCA / 2-AIA matrix;

[0046] Figure 10 MALDI mass spectra of oligosaccharide additives in different foods were obtained using CHCA / 2-AIA matrix. Figure 10 AC represents three different brands of juice samples; Figure 10 DF represents three different brands of milk samples; Figure 10 GI represents three different brands of yogurt samples. Detailed Implementation

[0047] The following embodiments will further illustrate the present invention, but the present invention is not limited to the following embodiments.

[0048] Unless otherwise specified, the samples, reagents, etc. used in the following examples are commercially available. The invention is described below through specific examples, but is not limited thereto.

[0049] The matrix-assisted laser desorption / ionization time-of-flight mass spectrometer used in the following embodiments is specifically model UlrafleXtreme. TM The MALDI-TOF / TOF MS (Bruker Daltonic, Germany) used a 355 nm Nd:YAG laser. All detections were performed in positive ion reflection mode. Positive ion reflection mode parameters: accelerating voltage, 25.00 kV; delayed extraction voltage, 22.30 kV; delayed extraction time, 130 ns; reflector voltage 1, 26.50 kV; reflector voltage 2, 13.50 kV; lens voltage, 7.50 kV; frequency, 1000 Hz. The target plate used was a 384 polished steel plate (MTP 384 polished steel), and mass spectrometry data analysis was performed using Bruker Flexanalysis 3.4 software.

[0050] The following abbreviations or foreign terms are used throughout this invention:

[0051] 2-AIA, 2-aminoisophthalic acid;

[0052] ACN, acetonitrile;

[0053] ACT, acetone;

[0054] CHCA, α-cyano-4-hydroxycinnamic acid;

[0055] ddH2O, deionized water;

[0056] D2000, oligodextrose 2000;

[0057] DHB, 2,5-dihydroxybenzoic acid;

[0058] DMSO, dimethyl sulfoxide;

[0059] ESI, electrospray mass spectrometry;

[0060] EtOH, ethanol;

[0061] fmol, femtomol;

[0062] G6, maltohexaose;

[0063] G7, maltoheptaose;

[0064] HRMS high-resolution mass spectrometry;

[0065] Hz, Hertz;

[0066] IS, internal standard;

[0067] LOD, limit of detection;

[0068] LOQ, Limit of Quantification;

[0069] MALDI, matrix-assisted laser desorption / ionization;

[0070] MeOH, methanol;

[0071] min, minutes;

[0072] MP, melting point;

[0073] MR, molar ratio;

[0074] MS, mass spectrometry;

[0075] MW, molecular weight;

[0076] m / z, charge-to-weight ratio;

[0077] NMR, nuclear magnetic resonance;

[0078] PA, proton affinity;

[0079] pKa, acidity coefficient;

[0080] R 2 The correlation coefficient of the quantitative curve;

[0081] RSD, Relative Standard Deviation;

[0082] THAP, 2,4,6-trihydroxyacetophenone;

[0083] THF, tetrahydrofuran;

[0084] rt, room temperature;

[0085] S / N, signal-to-noise ratio;

[0086] SA, sinapic acid;

[0087] SD, standard deviation;

[0088] μL;

[0089] V volts;

[0090] Example 1: Characterization of 2-AIA

[0091] 2-AIA was characterized using NMR, high-resolution ESI, ultraviolet absorption spectroscopy, and melting point apparatus.

[0092] In Example 1, Figure 1 For 2-AIA 1 1H NMR spectrum (1A) 13 The results of the 1C NMR spectrum (1B) and high-resolution ESI negative ion mode mass spectrum (1C) are as follows: 1 H NMR (400MHz, DMSO-d6) δ12.81 (s, 2H), δ8.20 (s, 2H), 8.02 (d, J = 8Hz, 2H), 7.71 (s, 1H), 6.57 (t, J = 8Hz, 1H); 13 C NMR (126MHz, DMSO-d6) δ 169.31, 152.89, 137.40, 113.40, 113.74; High-resolution ESI (m / z): 180.0308 [MH] - The spectral signal is clean and corresponds one-to-one with its structural information, indicating that the 2-AIA used in this embodiment is a high-purity compound obtained after separation and purification. Figure 2The table shows the UV absorption spectra of DHB and 2-AIA at the same molar concentration. It can be seen that although the UV absorption peak shapes of 2-AIA and DHB are basically similar, the maximum absorption peak of 2-AIA is red-shifted relative to DHB, and its absorption intensity at the MALDI laser (355 nm) is higher. Therefore, 2-AIA possesses the best optical performance for MALDI laser compatibility. Table 1 shows that the melting point of DHB is 195–196℃, and the melting point of 2-AIA is 312–315℃, indicating that 2-AIA meets the requirements of the high-vacuum working environment in mass spectrometry.

[0093] Example 2: 2-AIA itself as a MALDI matrix to detect oligosaccharides

[0094] (1) Prepare a 200 pmol / μL G6 oligosaccharide solution and store it in a refrigerator at 4℃;

[0095] (2) Prepare a 0.05 mol / L 2-AIA matrix solution in Example 1 using an equal volume mixture of acetone and dimethyl sulfoxide as a solvent. The solution can be stored in a refrigerator at 4°C.

[0096] (3) Prepare a 0.05 mol / L DHB methanol solution and store it in a refrigerator at 4℃;

[0097] (4) Take 1 μL of G6 sugar solution from step (1) and 1 μL of 2-AIA matrix solution from step (2) respectively, spot them using the dry drop method and let them air dry at 20℃;

[0098] (5) As a control, take 1 μL of G6 sugar solution from step (1) and 1 μL of 0.05 mol / L DHB methanol solution from step (2), spot them using the dry drop method and dry them naturally at 20℃.

[0099] (6) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0100] In Example 2, Figure 3 The MALDI mass spectra of G6 were detected using DHB and 2-AIA as matrices, respectively, where 1013.317 Da corresponds to [G6+Na]. + peak. Figure 4 The signal-to-noise ratio of B is significantly higher than that of B. Figure 4 A. As can be seen, 2-AIA itself performs better as a MALDI matrix than the traditional matrix DHB, making it an ideal matrix for detecting oligosaccharides.

[0101] Example 3: Construction of oligosaccharide analysis strategy based on CHCA / 2-AIA binary matrix

[0102] Using the 200 pmol / μL G6 oligosaccharide solution prepared in Example 2 as the model compound, the types of mixed solvents, the volume ratio of dimethyl sulfoxide, the concentration of 2-AIA, the spotting method, the drying temperature, the selection of traditional organic matrices, and the molar ratio of CHCA to 2-AIA in the binary matrix were investigated. The specific steps are as follows:

[0103] (1) Optimization of mixed solvents: Dimethyl sulfoxide (DMSO) was mixed in equal volumes with acetonitrile (ACN), acetone (ACT), ethanol (EtOH), methanol (MeOH), and tetrahydrofuran (THF) to obtain five different mixed solvents. 2-AIA matrix solutions with a concentration of 0.05 mol / L were prepared using each mixed solvent. 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of the 2-AIA matrix solutions prepared with different mixed solvents were taken, spotted using the dry-drop method, and allowed to air dry at 20°C before MALDI positive ion reflectance mode detection. Figure 4 As shown in Figure A, when ACN and DMSO are used as a mixed solvent (ACN / DMSO), the [G6+Na] measured in the 2-AIA matrix... + Ions have the highest signal-to-noise ratio;

[0104] (2) Optimization of DMSO volume ratio: Using ACN / DMSO as a mixed solvent, 2-AIA matrix solutions with a concentration of 0.05 mol / L were prepared at DMSO volume ratios of 1%, 5%, 10%, 15%, and 20%. 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of the 2-AIA matrix solutions prepared with different volume ratios of DMSO as the mixed solvent were taken, spotted using the dry-drop method, and allowed to air dry at 20°C before MALDI positive ion reflectance mode detection. Figure 4 As shown in Figure B, with the gradual increase of the proportion of DMSO in the mixed solvent, the [G6+Na] measured in the 2-AIA matrix... + The signal-to-noise ratio of the ions increased significantly and remained constant after the volume ratio of DMSO reached 15%. Taking into account the drying time, the best evaluation was achieved when the volume ratio of DMSO in the ACN / DMSO mixed solvent was 15%.

[0105] (3) Optimization of 2-AIA concentration: Using ACN / DMSO with a DMSO volume ratio of 15% as a mixed solvent, 2-AIA matrix solutions with concentrations of 0.005 mol / L, 0.01 mol / L, 0.02 mol / L, 0.04 mol / L, and 0.08 mol / L were prepared. 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of the 2-AIA matrix solutions of different concentrations were respectively spotted using the dry-drop method and allowed to air dry at 20°C before MALDI positive ion reflectance mode detection. Figure 4As shown in Figure C, with increasing 2-AIA matrix concentration, [G6+Na] + The signal-to-noise ratio of the ions showed a trend of first increasing and then decreasing, and the highest response value was measured when the 2-AIA concentration was 0.02 mol / L.

[0106] (4) Optimization of the spotting method: A 0.02 mol / L 2-AIA matrix solution was prepared using a 15% (v / v) ACN / DMSO mixed solvent. 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of the 2-AIA matrix solution were spotted sequentially onto the same well of the target plate. The mixture was then repeatedly pipetted 10 times onto the MALDI target plate to ensure homogeneity, and allowed to air dry at 20°C. 1 μL of the 2-AIA matrix solution was then spotted onto different wells of the same target plate. After air drying at 20°C, 1 μL of the G6 sugar solution prepared in Example 2 was spotted onto the upper layer of the matrix crystals. After air drying at 20°C, MALDI positive ion reflectance mode detection was performed. Figure 4 As shown in D, different spotting methods resulted in significant differences in the matrix properties of the 2-AIA matrix, and the signal-to-noise ratio of G6 was higher when the dry drop method was used for spotting.

[0107] (5) Optimization of drying temperature: Take 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of the 2-AIA matrix solution prepared in step (4), spot them using the dry drop method, and dry them at 20 and 65 °C respectively, then perform MALDI positive ion reflectance mode detection. Figure 4 As shown in E, drying temperature is also an important factor affecting the detection results, and room temperature drying is more conducive to obtaining a higher signal response.

[0108] (6) Selection of traditional organic matrices: Using the 2-AIA matrix solution prepared in step (4) as the solvent, and DHB, CHCA, SA, and THAP as solutes, four binary matrix solutions of DHB / 2-AIA, CHCA / 2-AIA, SA / 2-AIA, and THAP / 2-AIA with a solute concentration of 0.02 mol / L were prepared. 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of each binary matrix solution were taken, spotted using the dry-drop method, and allowed to dry naturally at 20°C before MALDI positive ion reflectance mode detection. Figure 5 MALDI mass spectra of G6 and the co-crystallization morphology of the corresponding matrix and G6 were detected for different binary matrices. It can be seen that the CHCA / 2-AIA binary matrix obtained by combining CHCA and 2-AIA can give full play to the synergistic effect of the two matrices, which can not only significantly improve the ionization efficiency of oligosaccharides, but also significantly improve the co-crystallization morphology of oligosaccharides and matrices.

[0109] (7) Optimization of the molar ratio of CHCA to 2-AIA in the binary matrix: Using the 2-AIA matrix solution prepared in step (4) as the solvent and CHCA as the solute, CHCA / 2-AIA binary matrix solutions with CHCA concentrations of 0.005 mol / L, 0.01 mol / L, 0.02 mol / L, 0.04 mol / L and 0.08 mol / L were prepared, i.e., CHCA / 2-AIA binary matrix solutions with molar ratios of 0.25, 0.5, 1, 2 and 4 respectively. 1 μL of the G6 sugar solution prepared in Example 2 and 1 μL of CHCA / 2-AIA binary matrix solutions with different molar ratios were taken, spotted using the dry drop method, and naturally dried at 20℃ before MALDI positive ion reflectance mode detection. Figure 6 The effect of different molar ratios of CHCA and 2-AIA on the detection of G6. Figure 6 As shown in Figure A, with the increase of the CHCA molar ratio, the assisted ionization efficiency of the CHCA / 2-AIA matrix shows a trend of first increasing and then decreasing, reaching a peak at a molar ratio of 2. This is the effect on the G6 signal intensity. (In summary...) Figure 6 The effect of the molar ratio of CHCA to 2-AIA in B on co-crystallization: when the molar ratio is equal to 2, the matrix and the analyte co-crystallize to exhibit a uniform and dense morphology.

[0110] Example 3 shows that by optimizing the sample preparation conditions of the CHCA / 2-AIA binary matrix, higher ionization efficiency and more uniform co-crystallization were achieved in oligosaccharide analysis. Based on the above optimization results, subsequent experimental conditions were performed using a mixed solvent of ACN / DMSO at a volume ratio of 15% (DMSO) to prepare a CHCA / 2-AIA binary matrix solution with a 2-AIA concentration of 0.02 mol / L and a CHCA to 2-AIA molar ratio of 2. The solution was spotted using the dry-drop method and allowed to air dry at 20°C before being detected in MALDI positive ion reflectance mode.

[0111] Example 4: Qualitative investigation of CHCA / 2-AIA binary matrix to detect oligosaccharides with different molecular weights

[0112] 1. CHCA / 2-AIA as a matrix for detecting oligosaccharides of different molecular weights

[0113] (1) Prepare a 200 pmol / μL G7 oligosaccharide solution, and then dilute it to 125 pmol / μL, 25 pmol / μL, 10 pmol / μL, 5 pmol / μL, 1 pmol / μL, 500 fmol / μL, 100 fmol / μL and 10 fmol / μL to obtain G7 sugar solutions of various concentrations, which can be stored in a refrigerator at 4℃;

[0114] (2) Prepare standard oligosaccharide solutions of NGA3, NA2 and Man-8 at 20 pmol / μL respectively, and then dilute them to 5 pmol / μL, 2 pmol / μL, 1 pmol / μL, 500 fmol / μL, 100 fmol / μL and 10 fmol / μL to obtain NGA3, NA2 and Man-8 sugar solutions of various gradient concentrations, which can be stored in a refrigerator at -20℃.

[0115] (3) Take 1 μL of the G7 oligosaccharide solution in step (1) or the standard oligosaccharide solution of NGA3, NA2 or Man-8 in step (2) and 1 μL of the CHCA / 2-AIA binary matrix solution in Example 3, spot the samples using the dry drop method and dry them naturally at 20°C.

[0116] (4) As a control, take 1 μL of the G7 oligosaccharide solution in step (1) or the standard oligosaccharide solution of NGA3, NA2 or Man-8 in step (2) and 1 μL of the 0.05 mol / L DHB methanol solution in Example 2, spot the samples using the dry drop method and dry them naturally at 20°C.

[0117] (5) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0118] In Example 4 Figure 7 The detection limits for oligosaccharides of different molecular weights were determined using DHB and CHCA / 2-AIA as MALDI matrices, respectively. Figure 7 A, C, E, and G are MALDI spectra detected using CHCA / 2-AIA as the matrix. Figure 7 B, D, F, and H are MALDI spectra obtained using DHB as the matrix. The specific detection limits are summarized in Table 1. When using DHB as the matrix to detect G7, NGA3, NA2, and Man-8, the detection limit was 500 fmol, while when using CHCA / 2-AIA as the matrix, the detection limit was as low as 10 fmol. This demonstrates that the oligosaccharide analysis strategy using the binary matrix CHCA / 2-AIA as the core exhibits excellent detection performance, enabling the detection of oligosaccharides with higher degrees of polymerization at lower concentrations. Furthermore, the detection sensitivity of this method is 50 times higher than that of the traditional DHB matrix.

[0119] Table 1. Proton affinity, melting point, and detection limits for oligosaccharides of different molecular weights of the matrix.

[0120]

[0121] 2. CHCA / 2-AIA as a matrix for detecting oligosaccharide mixtures

[0122] (1) Prepare a 0.5 mg / mL D2000 oligosaccharide solution and store it in a refrigerator at 4℃;

[0123] (2) Take 1 μL of D2000 sugar solution from step (1) and 1 μL of CHCA / 2-AIA binary matrix solution from Example 3, spot them using the dry drop method and let them dry naturally at 20°C;

[0124] (3) The target plate was sent into the MALDI mass spectrometer and data acquisition was performed using the positive ion reflection mode.

[0125] In Example 4, Figure 8 The image shows the MALDI mass spectrum of the oligosaccharide mixture D2000 detected using CHCA / 2-AIA as the MALDI matrix. It is evident that the CHCA / 2-AIA matrix exhibits a significant signal-to-noise ratio when detecting the dextran mixture, and each mass peak can be distinguished and assigned. Specifically, 1661.525 Da corresponds to [G10+Na]. – The peak at 1823.578 Da corresponds to [G11+Na]. – The peak, at 162 Da, corresponds to the molecular weight of a single sugar residue, and exhibits a standard normal distribution within the mass range of 500–3000 Da, covering most oligosaccharide chains. This demonstrates that the CHCA / 2-AIA binary matrix has broad applicability for oligosaccharide detection and is also suitable for the direct detection of oligosaccharide mixtures.

[0126] Example 5: Quantitative investigation of method stability and linear range of CHCA / 2-AIA binary matrix

[0127] (1) Prepare a G7 oligosaccharide solution with a concentration of 2000 fmol / 0.5 μL as an internal standard solution, which can be stored in a refrigerator at 4℃;

[0128] (2) Take the standard oligosaccharide solution of NGA3, NA2 and Man-8 with a concentration of 20 pmol / μL from Example 4, and dilute it to 10000 fmol / 0.5 μL, 5000 fmol / 0.5 μL, 2500 fmol / 0.5 μL, 1250 fmol / 0.5 μL and 625 fmol / 0.5 μL respectively to obtain gradient concentration sugar solutions of NGA3, NA2 and Man-8, which can be stored in a refrigerator at -20℃;

[0129] (3) Take 1 μL of G6 sugar solution from Example 2 and 1 μL of DHB matrix from Example 2 respectively, and spot them using the dry drop method. Then take 1 μL of G6 sugar solution from Example 2 and 1 μL of CHCA / 2-AIA binary matrix solution from Example 3 respectively, and spot them using the dry drop method on different wells of the same target plate. After the target plate is naturally dried at 20°C, it is sent to a MALDI mass spectrometer for detection in positive ion reflectance mode. Each well is sampled 45 times.

[0130] (4) Take 0.5 μL of NGA3 sugar solution of different concentrations in step (2), 0.5 μL of G7 internal standard solution in step (1) and 1 μL of CHCA / 2-AIA binary matrix solution in Example 3, spot them using the dry drop method and dry them naturally at 20℃, and then perform MALDI positive ion reflectance mode detection. Take three random samples from each spotting well and take the average value.

[0131] (5) Take 0.5 μL of NA2 sugar solution of different concentrations in step (2), 0.5 μL of G7 internal standard solution in step (1) and 1 μL of CHCA / 2-AIA binary matrix solution in Example 3, spot them using the dry drop method and dry them naturally at 20℃, and then perform MALDI positive ion reflectance mode detection. Take three random samples from each spotting well and take the average value.

[0132] (6) Take 0.5 μL of Man-8 sugar solution of different concentrations in step (2), 0.5 μL of G7 internal standard solution in step (1) and 1 μL of CHCA / 2-AIA binary matrix solution in Example 3, spot them using the dry drop method and dry them naturally at 20°C, and then perform MALDI positive ion reflectance mode detection. Take three random samples from each spotting well and take the average value.

[0133] In Example 5, Figure 9 A represents the signal reproducibility of oligosaccharide detection using DHB and CHCA / 2-AIA matrices, respectively. By averaging and dimensionlessly processing the signal intensities from 45 samples, it is clearly evident that the S / N ratio of the G6 ion detected using the CHCA / 2-AIA matrix exhibits very small fluctuations and remains consistently near the mean. Furthermore, the relative standard deviation (RSD) for G6 detection using the CHCA / 2-AIA matrix is ​​only 6.0%, significantly lower than the control group using DHB as the matrix (RSD = 71%). This demonstrates that the oligosaccharide analysis method based on the CHCA / 2-AIA binary matrix exhibits good stability and reproducibility, facilitating accurate quantitative analysis. Figure 9 BD investigated the linear range of three oligosaccharides, NGA3, NA2, and Man-8, using G7 as an internal standard in positive ion reflectance mode. It was found that all three oligosaccharides exhibited good linearity in the concentration range of 0.625–10 pmol / 0.5 μL, with R0. 2 >0.999. Therefore, using CHCA / 2-AIA as a matrix allows for accurate quantification of various oligosaccharides over a wider linear range.

[0134] Example 6: Characterization of CHCA / 2-AIA binary matrix to detect oligosaccharide additives in food

[0135] (1) Fruit juice, milk, and yogurt were used as test materials. Three different brands of each test material were selected as the source, for a total of nine different food samples. 20 μL of each food sample was taken, diluted tenfold with ultrapure water, and filtered through a 0.45 μm PTFE membrane to obtain the food sample solution. Three copies of each food sample solution were prepared in parallel and can be stored at 4℃.

[0136] (2) Take 1 μL of the food sample solution in step (1) and 1 μL of the CHCA / 2-AIA binary matrix solution in Example 3, spot them using the dry drop method and dry them naturally at 20°C;

[0137] (3) The target plate was sent into the MALDI mass spectrometer and data acquisition was performed using the positive ion reflection mode.

[0138] In Example 6, Figure 10 To characterize oligosaccharide additives in different food samples using CHCA / 2-AIA as a matrix; Figure 10 AC represents fruit juice samples from three different sources; Figure 10 DF represents milk samples from three different sources; Figure 10 GI represents yogurt samples from three different sources. The results indicate that a rapid and high-throughput analysis of commercially available food products can be performed directly using the CHCA / 2-AIA binary matrix. Figure 10 The ion signals measured in the AI ​​uniformly exhibited a uniform Gaussian distribution model, and the mass difference between adjacent peaks was 162 Da, indicating the presence of oligosaccharide additives. Evaluation was conducted at different parameter levels, summarized in Table 2, specifically including the lowest and highest degrees of polymerization (n) of the oligosaccharide additives. s-h ); np, the degree of polymerization of the most abundant sugar chain in the oligosaccharide additive (n p The signal-to-noise ratio (S / N) of the highest peak ion concentration. max Number-average molecular weight (M) n Weight-average molecular weight (M) w The results showed that the fingerprint spectra of oligosaccharide additives in different food samples differed significantly. As a high-throughput characterization method for oligosaccharide additives in food, this invention may provide evidence for controlling food safety in production and contribute important data support and theoretical guidance for the activity research and production optimization of oligosaccharide additives.

[0139] Table 2 Evaluation of parameters of oligosaccharide additives in different food samples

[0140]

[0141] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

The application of 1,2-aminoisophthalic acid as a MALDI matrix in the detection of oligosaccharide additives in food is characterized by, The oligosaccharide additive is G6 oligosaccharide. The application of 2,2-aminoisophthalic acid as a MALDI matrix combined with α-cyano-4-hydroxycinnamic acid as a binary matrix in the detection of oligosaccharide additives in food is characterized by, The oligosaccharide additive is G6 oligosaccharide.

3. The application according to claim 1 or 2, characterized in that, The 2-aminoisophthalic acid is as shown in formula (1): ； Equation (1).

4. The application according to claim 1, characterized in that, Specifically, it includes: (1) Reagent preparation 2-AIA is 2-aminoisophthalic acid, and a 2-AIA solution is prepared using dimethyl sulfoxide and acetone as a mixed solvent; CHCA is α-cyano-4-hydroxycinnamic acid, and a CHCA / 2-AIA binary matrix solution is prepared using CHCA as the solute and 2-AIA solution as the solvent; the sample solution is diluted with water. (2) Spotting Equal volumes of sample solution and CHCA / 2-AIA binary matrix solution were taken and spotted using the dry drop method or thin layer method to obtain the spotted target plate. (3) Drying After spotting, the target plate is placed until the mixed sample spots are completely dry, resulting in a dried target plate. (4) Mass spectrometry detection The dried target plate was sent into a MALDI-TOF-MS mass spectrometer and detected using positive ion reflectance mode.

5. The application according to claim 4, characterized in that, In step (1), the volume ratio of dimethyl sulfoxide in the mixed solvent of dimethyl sulfoxide and acetone is 12~18%.

6. The application according to claim 4, characterized in that, In step (1), the concentration of 2-AIA in the CHCA / 2-AIA binary matrix solution is 0.01~0.03 mol / L.

7. The application according to claim 4, characterized in that, In step (1), the molar ratio of CHCA to 2-AIA in the CHCA / 2-AIA binary matrix solution is 1.1~2.9:

1.

8. The application according to claim 4, characterized in that, In step (2), the sample solution is spotted in a volume of 0.5 to 1.2 μL. The spotting volume of the CHCA / 2-AIA binary matrix solution is 0.5~1.2 μL.

9. The application according to claim 4, characterized in that, In step (2), the dry drop method involves sequentially spotting the binary matrix solution and the sample solution onto the same spotting well on the target plate, and then using a pipette to repeatedly aspirate and tap the polished target plate 10 to 20 times to mix them. In step (2), the thin-layer method involves first spotting a binary matrix solution onto the sample well of the target plate, drying it, and then spotting the sample solution onto the upper layer of the matrix crystallization.

10. The application according to claim 4, characterized in that, In step (3), the target plate after spotting is placed at 20~25 ℃.

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