A method for synchronous extraction and determination of twelve anthocyanins based on l-carnitine-lactobionic acid-water deep eutectic solvent

Abstract

The present application relates to a kind of based on L-carnitine-lactobionic acid-water low eutectic solvent twelve anthocyanins synchronous extraction determination method, belong to food safety detection technical field.The twelve anthocyanins synchronous extraction and determination method of the present application, it is with L-carnitine, lactobionic acid and water constructs natural low eutectic solvent as anthocyanin extraction agent, utilize the acidic hydrogen bond network system formed by the quaternary ammonium type amphiphilic ion structure of L-carnitine, the polyhydroxy sugar acid structure of lactobionic acid and appropriate water, improve the synchronous release, extraction recovery rate and stability of different glycoside type anthocyanins in complex matrix, and combined with HPLC-MS / MS can simultaneously realize 12 kinds of structure similar anthocyanins Effective separation, simultaneous detection and accurate quantification;And method operation is simple, sensitivity is high, reproducibility is good, and the advantages such as wide applicable matrix range, can be used for the detection analysis and quality control of anthocyanin component in berry and its processing products.

Description

Technical Field

[0001] This invention relates to a method for the simultaneous extraction and determination of twelve anthocyanins based on an L-carnitine-lactobionic acid-water eutectic solvent, belonging to the field of food safety testing technology. Background Technology

[0002] Anthocyanins are a class of water-soluble pigments widely found in grapes, blueberries, mulberries, waxberries, purple cabbage, and various berries and grains. They belong to the flavonoid polyphenol compound family. They are not only the material basis for the red, purple, and blue-purple colors of fruits and vegetables, but also important indicators for evaluating varietal characteristics, maturity, processing quality, and storage stability, thus playing a significant role in food quality control.

[0003] Anthocyanins are typically formed by the combination of anthocyanins and glycosyl groups. Common anthocyanins include delphinidin, cyanidin, petunidin, pelargonidin, paeoniflorin, and malvidin. Among these, 3-O-glucosides and 3,5-O-diglucosides are representative types of anthocyanins found in berries and their processed products. 3-O-glucosides and their corresponding 3,5-O-diglucosides have similar core structures, but due to differences in the types of substituents (hydroxyl, methoxy, etc.) and the number and position of glycosyl groups, their molecular polarity, solubility, reversed-phase chromatography retention behavior, mass spectrometry ionization response, collision-induced fragmentation characteristics, and chemical stability all differ. Generally, 3,5-O-diglucosides, due to their larger number of glycosyl groups and stronger hydrophilicity, exhibit relatively weaker retention in reversed-phase chromatography systems, and their extraction efficiency, ionization response, and stability may also differ from their corresponding 3-O-glucosides. The aforementioned differences in structure and physicochemical properties make it challenging to achieve simultaneous extraction, effective separation, and accurate quantification of multiple anthocyanin monomers in the same analytical system.

[0004] Sample pretreatment is a crucial step affecting the accuracy and applicability of anthocyanin determination methods. Anthocyanins are highly sensitive to conditions such as pH, temperature, light, solvent composition, and extraction time, and are prone to degradation, transformation, or loss during extraction. Furthermore, samples from fresh fruit, juice, jelly, jam, and berry-derived health products vary significantly in terms of water content, sugars, organic acids, pectin, protein, polyphenols, other pigments, and excipient composition. Different matrices have inconsistent effects on target analyte release, solvent penetration, extraction recovery, chromatographic separation, and mass spectrometry ionization. In particular, 3-O-glucoside and 3,5-O-diglucoside may exhibit different recovery behaviors in the same pretreatment system due to differences in the number of glycosyl groups, polarity, and stability. Existing anthocyanin detection methods are mainly applicable to matrices such as fresh fruit, freeze-dried fruit, juice, or fruit juice beverages. In contrast, the applicability validation for the simultaneous determination of multiple anthocyanins in deeply processed or complex matrices such as jam, jelly, and berry-derived health products remains insufficient. Therefore, there is an urgent need to establish a pretreatment and detection method suitable for complex matrices of berries and their processed products, which takes into account the extraction efficiency, recovery balance and quantitative accuracy of various anthocyanins.

[0005] Currently, anthocyanin detection commonly employs ultraviolet-visible spectrophotometry, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). Ultraviolet-visible spectrophotometry is frequently used for determining total anthocyanin content due to its ease of operation; for example, Gu Huaishan et al. used this method to determine the total anthocyanin content in the fruit of the red ginseng variety. However, this method typically cannot effectively distinguish between structurally similar anthocyanin monomers. HPLC can be used for the separation and quantification of specific anthocyanin monomers. For instance, Xia Hui et al. used HPLC to determine the content of cyanidin-3-O-glucoside in black rice, achieving good linearity in the concentration range of 0.20–50.0 μg / mL, with a correlation coefficient R² of 0.999894. The limits of detection and quantitation were 0.1 mg / kg and 0.3 mg / kg, respectively. However, this type of method is often established for single or a few target components. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) offers high sensitivity and selectivity. For example, Sun Qiaoye et al. used this method to analyze and identify six anthocyanin components in "Qiufu" red raspberries, with cyanidin-3-glucoside having the highest content at 4.49 mg / g. This demonstrates that while existing detection methods have been applied to the determination of anthocyanins in some fruit, grain, or berry samples, their applications are primarily limited to raw materials or relatively simple matrices. The applicability and systematic validation of methods for the simultaneous determination of multiple anthocyanin monomers in deeply processed or complex matrices such as jams, jellies, and berry-derived health products remain insufficient.

[0006] Based on this, the present invention aims to solve the technical problems of insufficient greenness of existing traditional organic solvent extraction systems such as acidified methanol and acidified ethanol, uneven extraction and recovery rates of anthocyanins of different glycoside types in complex matrices, easy degradation of anthocyanins during extraction, and difficulty of achieving simultaneous extraction, effective separation and accurate quantification of six 3-O-glucoside anthocyanins and six 3,5-O-diglucoside anthocyanins in complex matrices such as various berries and their deep-processed products. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for the simultaneous extraction and determination of twelve anthocyanins based on a low-melting-molecular solvent of L-carnitine, lactobionic acid, and water. This method utilizes a natural low-melting-molecular solvent constructed from L-carnitine, lactobionic acid, and water as the anthocyanin extractant. It leverages the quaternary ammonium zwitterionic structure of L-carnitine, the polyhydroxy glycolic acid structure of lactobionic acid, and the acidic hydrogen bond network system formed by an appropriate amount of water to improve the simultaneous release, extraction recovery, and stability of different anthocyanin glycoside types in complex matrices. Combined with HPLC-MS / MS, it can simultaneously achieve the effective separation, simultaneous detection, and accurate quantification of 12 structurally similar anthocyanins. Furthermore, the method boasts advantages such as simple operation, high sensitivity, good repeatability, and a wide range of applicable matrices, making it suitable for the detection, analysis, and quality control of anthocyanin components in berries and their processed products.

[0008] To achieve the above objectives, the following technical solution is provided: This invention provides a method for the simultaneous extraction and determination of twelve anthocyanins using an L-carnitine-lactobionic acid-water eutectic solvent, the method comprising the following steps: (1) Sample extraction Weigh the sample, add L-carnitine-lactobionic acid-water natural eutectic solvent, mix well, and then perform vortexing and ultrasonic-assisted extraction; after extraction, centrifuge and take the supernatant; dilute the supernatant with methanol aqueous solution containing formic acid or acetonitrile aqueous solution containing formic acid, vortex mix well, centrifuge again, and filter the supernatant through a microporous membrane to obtain the sample extract to be tested. (2) Preparation of matrix standard solution Using the blank matrix extract obtained in the same way as in step (1), a series of standard solutions of 12 anthocyanin mixed standards were prepared. (3) Anthocyanin content determination The series of anthocyanin mixed standard solutions prepared in step (2) and the sample extract prepared in step (1) were detected by HPLC-MS / MS. A standard curve was established with the concentration of each target anthocyanin as the abscissa and the corresponding chromatographic peak area or peak area ratio as the ordinate. The content of the twelve anthocyanins in the sample was calculated based on the response value of each target substance in the sample extract.

[0009] In one embodiment, the twelve anthocyanins include delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin-3,5-O-diglucoside, cyanidin-3,5-O-diglucoside, petunidin-3,5-O-diglucoside, pelargonidin-3,5-O-diglucoside, paeoniflorin-3,5-O-diglucoside, and malvidin-3,5-O-diglucoside.

[0010] In one embodiment, the L-carnitine-lactobic acid-water natural eutectic solvent in step (1) is prepared by mixing L-carnitine, lactobionic acid and water in proportion, heating and stirring until a transparent and homogeneous liquid is formed, and then cooling to obtain the L-carnitine-lactobic acid-water natural eutectic solvent.

[0011] In one embodiment, in the L-carnitine-lactobic acid-water natural eutectic solvent, the molar ratio of L-carnitine to lactobionic acid is 1:(0.2~3.0), preferably 1:(0.5~2.0), and more preferably 1:(0.8~1.5).

[0012] In one embodiment, the water content in the L-carnitine-lactobionic acid-water natural eutectic solvent is 15-65 wt% of the total mass of the L-carnitine-lactobionic acid-water natural eutectic solvent, preferably 25-50 wt%, and more preferably 30-45 wt%.

[0013] In one embodiment, the pH of the L-carnitine-lactobionic acid-water natural eutectic solvent in step (1) is 1.5 to 4.0, preferably 2.0 to 3.2, and more preferably 2.3 to 2.8.

[0014] In one embodiment, the heating and stirring temperature is 30~90℃, preferably 40~70℃; the stirring time is 10~120 min, preferably 20~60 min.

[0015] In one embodiment, the mass-to-volume ratio of the sample in step (1) to the natural eutectic solvent L-carnitine-lactobionic acid-water is 1:(5~60) g / mL, preferably 1:(10~40) g / mL, and more preferably 1:(15~25) g / mL.

[0016] In one embodiment, the vortex oscillation conditions in step (1) are 1000~2000 r / min for 2~6 min, preferably 1000~1500 r / min for 3~5 min.

[0017] In one embodiment, the temperature of the ultrasonic-assisted extraction in step (1) is 20~55℃, preferably 30~45℃; the ultrasonic-assisted extraction time is 5~60 min, preferably 20~40 min; and the ultrasonic frequency is 40 kHz.

[0018] In one embodiment, the centrifugation conditions in step (1) are 4000~5500 r / min, 10~25 min, 4℃; the re-centrifugation conditions are 9000~11000 r / min, 2~8 min, 4℃.

[0019] In one embodiment, the solution used for dilution in step (1) is a methanol-water-formic acid system or an acetonitrile-water-formic acid system; wherein the volume fraction of methanol or acetonitrile is 30-70%, preferably 45-55%; and the volume fraction of formic acid is 0.5-3.0%, preferably 1.0%.

[0020] In one embodiment, the liquid chromatography conditions for HPLC-MS / MS detection in step (3) are as follows: column: ACQUITY-UPLC-BEH-C18 (100 mm × 2.1 mm, 1.7 μm), column temperature 35~45℃; flow rate: 0.3~1.5 mL / min; mobile phase: A is formic acid aqueous solution, B is acetonitrile; gradient elution, injection volume: 2~5 μL.

[0021] In one embodiment, the volume fraction of formic acid in the mobile phase A is 0.5-2.0%, preferably 1.0%.

[0022] In one embodiment, the segmented linear gradient elution program is as follows: 0-2 min, 5% acetonitrile phase; 2-10 min, 5%-30% acetonitrile phase; 10-15 min, 30%-80% acetonitrile phase; 15-18 min, 80% acetonitrile phase; 18-18.5 min, 80%-5% acetonitrile phase; 18.5-20 min, 5% acetonitrile phase.

[0023] In one embodiment, the mass spectrometry conditions in step (3) are: using an electrospray ionization source, positive ion detection mode, and multiple reaction monitoring mode for qualitative and quantitative analysis.

[0024] In one embodiment, the mass spectrometry conditions for the HPLC-MS / MS determination in step (3) are as follows: an electrospray ionization source is used, positive ion detection mode is used, and multiple reaction monitoring mode is used; the curtain gas CUR is 20~30 psi, the collision gas CAD is Medium, the ion spray voltage IS is 5400~5600 V, the ion source temperature TEM is 450~550℃, the ion source gas 1 GS1 is 55~65 psi, and the ion source gas 2 GS2 is 35~45 psi.

[0025] Another object of the present invention is to provide the application of the above method in the analysis of anthocyanin composition, quality evaluation or the establishment of detection methods in berries and their processed products.

[0026] Beneficial effects: This invention establishes a method for the simultaneous extraction and HPLC-MS / MS determination of twelve anthocyanins using a natural eutectic solvent of L-carnitine-lactobionic acid-water. Compared with existing methods that mostly focus on the detection of total anthocyanin content, a few representative anthocyanin monomers, or specific matrix samples, this invention can simultaneously cover six 3-O-glucosides and six 3,5-O-diglucosides, providing a more comprehensive range of target analytes and enabling the simultaneous detection and quantification of multiple structurally similar anthocyanins. Specific advantages are as follows: (1) This invention uses L-carnitine-lactobic acid-water as a natural eutectic solvent for anthocyanin extraction, which differs from traditional organic solvent extraction systems such as acidified methanol and acidified ethanol. In the natural eutectic solvent, L-carnitine has a quaternary ammonium zwitterionic structure, and lactobionic acid has a polyhydroxy glycolic acid structure. Together with an appropriate amount of water, they form a solvent system with strong polarity, acidic microenvironment, and multiple hydrogen bonding interactions. This system can enhance the solvation effect on the anthocyanin glycoside structure, promote the release of anthocyanins from complex matrices, and help maintain the stability of anthocyanins during the extraction process. (2) This invention addresses the differences in polarity, solubility, stability, and extraction and recovery behavior among six types of 3-O-glucoside anthocyanins and six types of 3,5-O-diglucoside anthocyanins. By optimizing the molar ratio of L-carnitine to lactobionic acid, water content, mass-volume ratio of sample to natural eutectic solvent, extraction temperature, extraction time, and ultrasonic-assisted conditions, this invention enables anthocyanins of different structural types to achieve better extraction results. This method avoids the problem of uneven recovery rates of low-content components, sensitive components, or different glycoside types that may occur when existing methods only use total anthocyanin extraction yield or a single high-content component as optimization indicators. This method is beneficial to improving the accuracy and reliability of simultaneous determination of twelve target anthocyanins. (3) This invention combines L-carnitine-lactobionic acid-water natural eutectic solvent extraction with HPLC-MS / MS quantitative detection, and improves the compatibility of the natural eutectic solvent extract with the liquid chromatography-tandem mass spectrometry system through dilution, centrifugation and filtration steps. This method maintains a simple pretreatment process, short extraction time and convenient operation, while reducing the use of traditional acidified organic solvents in the extraction step, and is suitable for batch sample detection.

[0027] (4) The method described in this invention has good matrix adaptability, extraction recovery rate, process efficiency, precision and accuracy. It can be applied to the simultaneous determination of twelve anthocyanins in various berry matrices and their processed products, such as fresh fruit, juice, jam, jelly and berry-derived health products. It can provide a reliable technical means for the anthocyanin composition analysis, quality evaluation and quality control of berries and their processed products. Attached Figure Description

[0028] Figure 1 The ion current chromatograms of the six 3-O-glucan anthocyanins of the present invention at a concentration of 10 ng / ml are shown below. Figure 2 The ion current chromatograms of the six 3,5-O-diglucosides of the present invention at a concentration of 10 ng / ml are shown. Figure 3 This is a graph showing the effect of different extractants on the recovery rate of 12 anthocyanins in Comparative Example 1 of this invention; Figure 4 This is a graph showing the effect of different molar ratios on the recovery rates of 12 anthocyanins in Comparative Example 2 of this invention; Figure 5 This is a graph showing the effect of water content on the recovery rate of 12 anthocyanins in Comparative Example 2 of this invention; Figure 6 This is a graph showing the effect of different ultrasound times on the recovery rates of 12 anthocyanins in Comparative Example 3 of this invention. Figure 7 This is a graph showing the effect of different extraction temperatures on the recovery rates of 12 anthocyanins in Comparative Example 3 of this invention; Figure 8 This is a graph showing the effect of different extractants on the stability of 12 anthocyanins in Comparative Example 4 of this invention; Figure 9 This is a comparative ion flow chromatogram of Comparative Example 5 of the present invention; Figure 10 This is a data graph illustrating the verification of the method in Comparative Example 5 of the present invention; Figure 11 This is a data graph illustrating the verification of the method in Comparative Example 6 of the present invention. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0030] 1. Instruments, reagents and materials ExionLC™ QTRAP 5500+ high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS, SCIEX, USA); BL-2000F electronic balance (Setra, USA); Heraeus Multifuge X1R high-speed refrigerated centrifuge (Ningbo Noli Electronic Technology Co., Ltd.); SB25-12DT ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); Multi Reax automatic vortex mixer (Heidolph, Germany); thermostatic magnetic stirrer (Qingdao Juchuang Century Environmental Protection Technology Co., Ltd.); pH meter (Shanghai Yitian Scientific Instruments Co., Ltd.); rotational viscometer (Beijing Delit Technology Co., Ltd.); Milli-Q ultrapure water system (Supelco, Wuxi).

[0031] Acetonitrile and methanol, chromatographic grade reagents, were purchased from Supelco, USA; formic acid, chromatographic grade reagent, was purchased from Anaqua Chemicals Supply, USA; L-carnitine, purity ≥98.0%, was purchased from Shanghai Anpu Experimental Technology Co., Ltd.; lactobionic acid, purity ≥98.0%, was purchased from Beijing Innocare Technology Co., Ltd.; and ultrapure water was used for the experiment. L-carnitine, lactobionic acid, and water were used to prepare a natural eutectic solvent of L-carnitine-lactobic acid-water, which served as the anthocyanin extractant in the sample pretreatment process of this invention.

[0032] Twelve anthocyanin single-standard reference materials: petunia-3,5-O-diglucoside (Pt3,5G) standard, purchased from Shanghai OriLeaf Company; petunia-3-O-glucoside (Pt3G), pelargonidin-3,5-O-diglucoside (Pg3,5G) standard, purchased from Beijing Manhage Company; delphinidin-3-O-glucoside (Del3G), cyanidin-3-O-glucoside (Cy3G), pelargonidin-3-O-glucoside (Pg3G), paeoniflorin-3-O-glucoside (Pn3G), malvain-3-O-glucoside (Mal3G), delphinidin-3,5-O-diglucoside (Del3,5G), cyanidin-3,5-O-diglucoside (Cy3,5G), paeoniflorin- 3,5-O-diglucoside (Pn3,5G) and malvidin-3,5-O-diglucoside (Mal3,5G) standards, both with a purity greater than 95%, were purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0033] The microporous filter membrane is a 0.22 μm PTFE filter membrane or other filter membranes suitable for filtering sample extracts containing organic solvents; centrifuge tubes, brown sample vials, and pipette tips are all standard laboratory consumables.

[0034] 2. Preparation of standard solutions Twelve anthocyanin standard stock solutions: A methanol-formic acid mixture (95:5, v / v) was used as the blank matrix solution. Cy3G, Pg3G, Pn3G, Pt3G, Mal3G, Del3G, Cy3,5G, Pg3,5G, Pn3,5G, Pt3,5G, Mal3,5G, and Del3,5G were prepared into single standard stock solutions with mass concentrations of 2000, 1000, 500, 500, 500, 1000, 1000, 500, 500, 100, 1000, and 500 μg / mL, respectively, and then stored at -22°C.

[0035] Mixed standard working solution of 12 anthocyanins: Pipette 5 μL Cy3G, 10 μL Del3G, Pg3G, Cy3,5G and Mal3,5G, 20 μL Pn3G, Pt3G, Mal3G, Pg3,5G, Pn3,5G and Del3,5G, and 100 μL Pt3,5G standard stock solution into a brown sample vial. Add 735 μL of 50% methanol-water solution containing 1% formic acid to prepare a 10 μg / mL standard solution. Prepare fresh before use.

[0036] Preparation of L-carnitine-lactobionic acid-water natural eutectic solvent: Weigh L-carnitine and lactobionic acid, mix them at a molar ratio of 1:1, add 40 wt% of ultrapure water, heat and stir at 60°C until a transparent, homogeneous liquid without obvious crystallization is formed, and cool to room temperature to obtain the L-carnitine-lactobionic acid-water natural eutectic solvent.

[0037] Example 1 A method for the simultaneous extraction and detection of twelve anthocyanins in berries and their processed products using a natural eutectic solvent of L-carnitine-lactobionic acid-water comprises the following steps: (1) Sample pretreatment Blank sample: Accurately weigh 1 g of blank matrix sample into a 50 mL centrifuge tube, and add 7.5 μL Cy3G standard stock solution, 15 μL Del3G, Pg3G, Cy3,5G, Mal3,5G standard stock solutions, 30 μL Pn3G, Pt3G, Mal3G, Pg3,5G, Pn3,5G, Del3,5G standard stock solutions and 150 μL Pt3,5G standard stock solution in sequence. Mix well and let stand in the dark to allow the standard to fully contact the sample matrix. According to the mass-to-volume ratio of sample to L-carnitine-lactobionic acid-water natural eutectic solvent of 1:15 (g:mL), 15 mL of L-carnitine-lactobionic acid-water natural eutectic solvent was added to 1 g of the above blank matrix sample and the sample to be tested, respectively. The mixture was vortexed at 1000 r / min for 3 min, followed by ultrasonic-assisted extraction for 30-35 min at 35-40℃ and ultrasonic frequency of 40 kHz. The extraction was carried out in the dark. After extraction, the sample was centrifuged at 4℃ and 4500 r / min for 15 min, and the supernatant was collected. Transfer 1 mL of the supernatant to a new centrifuge tube, add 9 mL of 50% methanol aqueous solution containing 1% formic acid for dilution, mix well, vortex at 1000 r / min for 3 min, and then centrifuge at 4℃ and 10000 r / min for 3 min. Take the supernatant and filter it through a 0.22 μm PTFE filter membrane to obtain the spiked extract of the blank matrix to be tested, which is used for HPLC-MS / MS detection. The HPLC-MS / MS liquid chromatography conditions are as follows: The chromatographic column was an ACQUITY-UPLC-BEH-C18 column with dimensions of 100 mm × 2.1 mm and a diameter of 1.7 μm; the column temperature was 40℃; the flow rate was 0.5 mL / min; mobile phase A was 1% formic acid aqueous solution, and mobile phase B was acetonitrile; segmented linear gradient elution was used, and the elution program is shown in Table 1; the injection volume was 2 μL.

[0038] Table 1 Gradient elution program

[0039] The mass spectrometry conditions for HPLC-MS / MS are as follows: An electrospray ionization source was used, with positive ion detection mode and multiple reaction monitoring mode. The curtain gas CUR was 20 psi, the collision gas CAD was Medium, the ion spray voltage IS was 5500 V, the ion source temperature TEM was 500℃, the ion source gas 1GS1 was 60 psi, and the ion source gas 2GS2 was 40 psi. Table 2 provides basic information, parent ion information, retention time, and collision energy for 12 anthocyanins.

[0040] Table 2. Mass spectrometry data for qualitative and quantitative analysis of 12 anthocyanins

[0041] (2) Matrix effect Matrix effect (ME) is the influence caused by the competitive ionization of the target compound and co-extracted interfering substances in the matrix. For complex plant matrix samples such as berries and their processed products, co-extracted sugars, organic acids, phenolic acids, polyphenols, pigments, and other endogenous components may co-enter the mass spectrometry system with the target anthocyanin, affecting the ionization efficiency of anthocyanin in the electrospray ionization source, causing signal enhancement or suppression, thus affecting the accuracy of the detection results. Generally, ME=1.0 indicates no matrix effect; 0.8 < ME < 1.2 indicates a weak matrix effect, which can generally be ignored; 0.5 < ME < 0.8 or 1.2 < ME < 1.5 indicates a moderate matrix effect; and ME < 0.5 or ME > 1.5 indicates a strong matrix effect. When ME is between 0.8 and 1.2, the matrix effect is essentially negligible.

[0042] Matrix-matched calibration solutions and standard solutions of the same concentration were prepared using blank matrix extract and pure solvent. The matrix effect was represented by the ratio of the average peak area response of the negative sample (B) to the average peak area response of the pure solvent (A), calculated using the following formula:

[0043] This invention uses blank matrix samples to evaluate matrix effects. The results show that the matrix inhibitory concentrations (MEs) of the 12 anthocyanins range from 0.83 to 1.18, and all 12 anthocyanins exhibit moderate matrix inhibition effects. Considering that matrix effects cannot be completely eliminated, appropriate measures can only be taken to reduce their impact on compound analysis.

[0044] Therefore, this invention uses a blank matrix extract to prepare a standard curve to eliminate matrix effects to a certain extent. Furthermore, the matrix effects corresponding to different types of samples were also investigated. The results showed that after pretreatment, the matrix effects of different types of samples were not significantly different, all exhibiting weak matrix effects. Specific results are shown in Table 3.

[0045] Table 3. Matrix effects of 12 anthocyanins in different matrices

[0046] (3) Determination of the linear range of the method and the detection limit and quantitation limit. Seven matrix-matched mixed standard working solutions with gradient levels (10, 20, 50, 100, 200, 500, and 1000 μg / L) were prepared using a blank matrix. Quantitative working curves were plotted with concentration (x, μg / L) on the x-axis and peak area (y) of each target analyte on the y-axis. The correlation coefficients R0 of the linear regression equations for the 12 anthocyanin substances were obtained. 2 The values ​​ranged from 0.9968 to 0.9999, indicating a good linear relationship within the range of 10–1000 μg / L. The limits of detection (LOD) calculated using a signal-to-noise ratio (S / N) of 3 were 0.004–0.006 μg / mL, and the limits of quantitation (LOQ) were 0.012–0.018 μg / mL. Detailed results are shown in Table 4.

[0047] Table 4. Linear equations, linear ranges, limits of detection, and limits of quantification for 12 anthocyanins.

[0048] (4) Determination of the content of 12 anthocyanins in the sample to be tested The peak areas of each target analyte in the sample were substituted into the linear equation constructed above to calculate the content of 12 anthocyanins in the sample.

[0049] Example 2: Determination of Recovery and Precision Three different concentrations of anthocyanin standard solutions were added to the blank sample matrix solution. The concentrations were determined to be 1, 2, and 10 times the limit of quantitation (LOQ) of each anthocyanin (low: 0.012 / 0.018 μg / mL; medium: 0.024 / 0.036 μg / mL; high: 0.120 / 0.180 μg / mL). The assay was performed according to the method described in the examples. Each spiked level was tested in both intraday (6 replicates) and interday (3 consecutive days, 3 replicates per day). The results are shown in Table 5. The recoveries of each compound ranged from 79.71% to 95.03%, the intraday precision (RSDr) ranged from 0.8% to 4.4%, and the interday precision (RSDwR) ranged from 4.1% to 9.4%. All results met the methodological requirements.

[0050] Table 5. Recovery rates and precision of 12 anthocyanins

[0051] Example 3: Actual Sample Testing To verify the effectiveness of this method in actual samples, different types of samples were tested according to the method in Example 1. The samples included Xiahei grapes, grape juice, blueberry jam, plum-flavored jelly, fish oil anthocyanin health products, etc.

[0052] The results showed that nine anthocyanins were detected in the skin of Summer Black grapes, with the following specific categories and contents: Cy3G 18.44 mg / kg, Pn3G 26.65 mg / kg, Pt3G 674.64 mg / kg, Mal3G 265.87 mg / kg, Del3G 86.83 mg / kg, Cy3,5G 4.37 mg / kg, Pn3,5G 47.14 mg / kg, Pt3,5G 4.27 mg / kg, and Mal3,5G 84.17 mg / kg. Five anthocyanins were detected in the blueberry jam, with the specific categories and contents being Cy3G 9.36 mg / kg, Pn3G 3.55 mg / kg, Pt3G 29.19 mg / kg, Mal3G 72.71 mg / kg, and Del3G 72.02 mg / kg. Four anthocyanins were detected in the grape juice sample, with the specific types and contents being Cy3G 9.08 mg / kg, Pn3G 144.78 mg / kg, Pt3G 5.07 mg / kg, and Mal3G 6.42 mg / kg. Seven anthocyanins were detected in the plum-flavored jelly sample, with the following specific categories and contents: Cy3G 10.36 mg / kg, Pg3G 14.73 mg / kg, Pt3G 54.31 mg / kg, Mal3G 25.06 mg / kg, Del3G 27.61 mg / kg, Cy3,5G 6.44 mg / kg, and Pg3,5G 7.08 mg / kg. Ten anthocyanins were detected in fish oil anthocyanin health products. The specific categories and contents were Cy3G 190.65mg / kg, Pg3G 24.61mg / kg, Pn3G 64.19mg / kg, Pt3G 35.34mg / kg, Mal3G 67.07mg / kg, Del3G 104.68mg / kg, Cy3,5G 24.71mg / kg, Pn3,5G 17.06mg / kg, Pt3,5G 7.39mg / kg, and Mal3,5G 43.62mg / kg. There were significant differences in the composition and content of anthocyanins among different varieties.

[0053] The above results indicate that this method is applicable to the simultaneous detection of twelve anthocyanins in actual berries and their processed products, and can provide technical support for the anthocyanin composition analysis and quality evaluation of berries and their processed products.

[0054] Comparative Example 1: Effect of different extractants on the recovery rate of twelve anthocyanins To compare the effects of different extractants on the simultaneous extraction of twelve anthocyanins at a concentration of 100 ng / ml, acidified aqueous solution (A), acidified methanol aqueous solution (B), choline chloride-lactic acid-water natural eutectic solvent (C), betaine-lactic acid-water natural eutectic solvent (D), and L-carnitine-lactobic acid-water natural eutectic solvent (E) were used as extractants. Each group used the same batch of blank matrix for spiked samples, and maintained consistent sample weighing, spiking level, solid-liquid ratio, vortexing conditions, ultrasonic time, ultrasonic temperature, centrifugation conditions, dilution factor, and HPLC-MS / MS detection conditions. After extraction, the spiked recoveries of the twelve anthocyanins were determined to evaluate the simultaneous extraction capabilities of different extractants.

[0055] The results are as follows Figure 3As shown, there are significant differences in the extraction recoveries of the twelve anthocyanins by different extractants. The acidified aqueous solution group showed some extraction ability for some anthocyanins, but the overall recovery rate of the twelve anthocyanins was relatively low, indicating that relying solely on an acidic aqueous environment is insufficient to fully extract anthocyanins with different polarities and degrees of glycosylation. The acidified methanol aqueous solution group showed improved extraction efficiency compared to the acidified aqueous solution group, but the recovery rates of some target anthocyanins remained relatively low, and the recovery rates varied considerably among the twelve anthocyanins. The choline chloride-lactic acid-water natural eutectic solvent group and the betaine-lactic acid-water natural eutectic solvent group showed good extraction effects on most anthocyanins, but there were still cases of insufficient recovery rates for some target compounds or uneven recovery of anthocyanins with different structures. In contrast, the L-carnitine-lactobic acid-water natural eutectic solvent group of this invention achieved high and relatively stable recoveries of all twelve anthocyanins, with an average recovery rate of 90.1414% and a minimum monomer recovery rate of 87.901%. The recoveries of all twelve target compounds were within the range of 87.901%-96.901%, demonstrating superior overall extraction performance compared to the other four extraction systems. These results indicate that the L-carnitine-lactobic acid-water natural eutectic solvent of this invention can effectively balance the simultaneous extraction of anthocyanins with different polarities and glycosylation types, exhibiting high overall recovery and good recovery uniformity, making it a preferred extractant for the simultaneous extraction of twelve anthocyanins.

[0056] Comparative Example 2: Effects of the molar ratio of L-carnitine to lactobionic acid and water content on the recovery rates of twelve anthocyanins The effects of the molar ratio of L-carnitine to lactobionic acid and water content on the properties of natural eutectic solvents (L-carnitine-lacobionic acid-water) and the extraction efficiency of anthocyanins were investigated. Natural eutectic solvents with different compositions were prepared, with L-carnitine to lactobionic acid molar ratios of 1:0.5, 1:1, 1:1.5, 1:2, and 1:3, and water contents of 20 wt%, 30 wt%, 40 wt%, 50 wt%, and 60 wt% of the total solvent mass. Each solvent group was used for ultrasonic-assisted extraction of twelve anthocyanins at a concentration of 100 ng / ml, following the method described in Example 1. The transparency, flowability, pH, viscosity, average recovery rate of the twelve anthocyanins, average recovery rate of 3-O-glucoside anthocyanins, average recovery rate of 3,5-O-diglucoside anthocyanins, and lowest recovery rate of a single anthocyanin were compared.

[0057] The results are as follows Figure 4 , 5As shown, the molar ratio of L-carnitine to lactobionic acid and the water content significantly affect the formation state, viscosity, and extraction efficiency of the natural eutectic solvent. When the proportion of lactobionic acid is too low, the system's acidity and hydrogen bond donor capacity are insufficient, which is not conducive to the stable existence and complete dissolution of anthocyanins. When the proportion of lactobionic acid is too high, the system viscosity increases, the mass transfer efficiency decreases, and the recovery rate of some anthocyanins decreases. The addition of an appropriate amount of water can reduce the viscosity of the natural eutectic solvent, improve sample wetting and mass transfer processes, thereby improving the anthocyanin extraction efficiency. However, when the water content is too high, the hydrogen bond network structure of the natural eutectic solvent is weakened, and the extraction selectivity and stabilizing protection effect decrease. Comparing the extraction recovery rate, recovery uniformity, and solvent flowability of different formulations, the system with a molar ratio of L-carnitine to lactobionic acid of 1:(0.8~1.5) and a water content of 30~45 wt% shows the best overall effect. Among them, a natural eutectic solvent of L-carnitine-lactobionic acid-water with a molar ratio of L-carnitine to lactobionic acid of 1:1 and a water content of 40 wt% can be used as a preferred method.

[0058] Comparative Example 3: Effects of ultrasound time and ultrasound temperature on the extraction efficiency of L-carnitine-lactobic acid-water natural eutectic solvent Following the method of Example 1, the effect of ultrasonic conditions on the extraction of twelve anthocyanins at a concentration of 100 ng / ml using L-carnitine-lactobiculic acid-water natural eutectic solvent was investigated. The ultrasonic time was set to 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, and 50 min; the ultrasonic temperature was set to 20℃, 30℃, 40℃, 50℃, and 60℃.

[0059] The results are as follows Figure 6 As shown, when the ultrasonic time is short, anthocyanins are not fully released from the sample, and the overall recovery rate of each target anthocyanin is low. With increasing ultrasonic time, the recovery rates of the twelve anthocyanins gradually increase, reaching an optimal level around 35 min. Further extending the ultrasonic time, the recovery rates of some heat-sensitive or less stable anthocyanins no longer increase significantly, and may even decrease, indicating that excessively long ultrasonic times may lead to structural instability or degradation of some anthocyanins. Ultrasonic temperature also affects the extraction efficiency of the natural eutectic solvent L-carnitine-lactobicacrylic acid-water.

[0060] like Figure 7As shown, at lower temperatures, the viscosity of the natural eutectic solvent is high, resulting in insufficient mass transfer efficiency and incomplete anthocyanin dissolution. Appropriately increasing the temperature can reduce the system viscosity and promote anthocyanin release from the sample. When the temperature continues to rise, the stability of some anthocyanins decreases, especially delphinidin, pelargonidin, and some diglucoside anthocyanins, which are more easily affected. Considering both extraction efficiency and anthocyanin stability, the preferred ultrasonic time is 20–45 min, more preferably 30–40 min; the preferred ultrasonic temperature is 30–45 °C, more preferably 35–40 °C.

[0061] Comparative Example 4: Effects of different extractants on the storage stability of twelve anthocyanins To investigate the stabilizing and protective effects of different extractants on twelve anthocyanins at a concentration of 100 ng / ml during storage, acidified aqueous solution (A), acidified methanol aqueous solution (B), choline chloride-lactic acid-water natural eutectic solvent (C), betaine-lactic acid-water natural eutectic solvent (D), and L-carnitine-lactobic acid-water natural eutectic solvent (E) were used as extractants for each group. The same sample, same solid-liquid ratio, and same ultrasonic-assisted extraction conditions were used for each group. After extraction, the samples were centrifuged, and the supernatant was collected from each group. The extracts from each group were placed into identical brown sealed sample bottles, with consistent liquid volume and headspace volume, and stored at 4°C in the dark. Samples were taken at 0 h, 12 h, 24 h, 72 h, 120 h, and 168 h of storage. After the same dilution, centrifugation, and filtration, the contents of the twelve anthocyanins were determined by HPLC-MS / MS according to the method in Example 1. At least three parallel samples were prepared for each extractant and each storage time point. Since the initial extraction capacity of different extractants for anthocyanins varies, the concentration of each anthocyanin in each extract at 0 h was used as the initial concentration, and the retention rate of the corresponding anthocyanin during storage was calculated using the following formula: Anthocyanin retention rate (%) = Anthocyanin concentration at the corresponding storage time point / Anthocyanin concentration at 0 h × 100% The average retention rates of twelve anthocyanins were calculated, and the storage stability protection effects of different extractants on anthocyanins with different structural types were evaluated.

[0062] The results are as follows Figure 8As shown, the average relative retention rates of the twelve anthocyanins in each extraction system gradually decreased with prolonged storage time, but the stabilizing and protective effects of different extractants varied significantly. The acidified aqueous solution group (A) showed the fastest decline, with average relative retention rates of approximately 72%, 55%, and 34% after 12 h, 24 h, and 168 h of storage, respectively, indicating that the acidified aqueous solution alone had limited stabilizing and protective effect on anthocyanins. The acidified methanol aqueous solution group (B) showed slightly better stability than group A, with retention rates of approximately 81%, 62%, and 38% after 12 h, 24 h, and 168 h of storage, respectively, but still showed a significant decline with prolonged storage time. The choline chloride-lactic acid-water natural eutectic solvent group (C) had higher average relative retention rates than groups A and B at all time points, with retention rates of approximately 59%, 53%, and 50% after 72 h, 120 h, and 168 h of storage, respectively, indicating that it had a certain stabilizing and protective effect on anthocyanins. The protective effect of the betaine-lactic acid-water natural eutectic solvent group (D) was further improved, with retention rates of approximately 63%, 58%, and 54% after 72 h, 120 h, and 168 h, respectively, which were generally higher than those of groups A, B, and C. The L-carnitine-lactobic acid-water natural eutectic solvent group (E) of this invention exhibited the highest average relative retention rate throughout the entire storage process. After 12 h, 24 h, and 72 h of storage, the retention rates were approximately 93%, 87%, and 79%, respectively, and after 120 h and 168 h, they remained at approximately 60% and 56%, respectively, all higher than other comparative groups. These results indicate that the natural eutectic solvent described in this invention can more effectively slow down anthocyanin degradation and reduce anthocyanin loss during post-extraction storage, providing good stabilizing protection for the twelve anthocyanins. Therefore, this solvent is not only suitable for the simultaneous extraction of the twelve anthocyanins but also superior to other comparative extraction systems in maintaining the storage stability of anthocyanins in the extract.

[0063] Comparison of Comparative Example 5 with the method disclosed in Chinese Patent Application CN111999411A The pretreatment and detection method disclosed in Chinese patent application CN111999411A was used as Patent A, and the detection effects of the method in the embodiments of this invention on twelve anthocyanins in different matrix samples were compared. The results are as follows: Figures 9-10 As shown.

[0064] Depend on Figure 9As can be seen, when using the liquid chromatography method described in Patent A to detect anthocyanin components, Pg3G, Pg3,5G, Del3G, and Del3,5G all exhibit abnormal chromatographic responses within their corresponding retention time ranges. These responses are characterized by irregular peak shapes, peak splitting, prominent shoulder peaks, or overlapping response signals, making it difficult to obtain clear integration boundaries for the target peak. Because these chromatographic peaks do not meet the requirements for good peak shape and effective separation, the peak areas obtained using this method are easily affected by interference signals and integration methods, and cannot accurately characterize the actual content of the aforementioned anthocyanins. Therefore, the mobile phase conditions and liquid chromatography method described in Patent A are insufficiently applicable for the accurate quantification of some 3-O-glucosides and 3,5-O-diglucosides of anthocyanins.

[0065] Depend on Figure 10 It is known that method A of patent has a certain detection effect on some matrices and some anthocyanins, but its recovery rate is greatly affected by the type of matrix and the structure of anthocyanins, and the overall fluctuation is obvious. In particular, when using method A of patent, anthocyanin components such as Pg3G, Pt3G, Del3G, Pg3,5G and Del3,5G have problems such as abnormal peak interference, irregular peak shape or unclear integration boundary, which makes it impossible to accurately integrate their peak areas, and thus cannot obtain reliable recovery rate determination results. Therefore, the data of the above anthocyanin components are missing or unreliable, and are not included in the effective recovery rate comparison analysis with the method of the embodiments of the present invention.

[0066] When a 70% recovery rate is used as the acceptable threshold, the patented method A exhibits recovery rates below 70% in some complex matrix samples. For example, in fish oil matrices, the recovery rates of anthocyanins such as Cy3G, Pn3G, Cy3,5G, and Pt3,5G are below 70%; in jelly matrices, the recovery rates of anthocyanins such as Cy3,5G are below 70%. These results indicate that the patented method A is insufficiently adaptable to complex matrices and some anthocyanins with poor stability or significant polarity differences, making it difficult to guarantee stable and acceptable recovery rates for all target anthocyanins. In contrast, this invention uses L-carnitine-lactobic acid-water as a natural eutectic solvent as the extraction agent. This extractant, through the quaternary ammonium zwitterionic structure of L-carnitine, the polyhydroxy glycolic acid structure of lactobionic acid, and an appropriate amount of water, forms an acidic hydrogen bond network system, which enhances the solvation of anthocyanin glycoside structures and improves the simultaneous release and stability of different glycoside types of anthocyanins in complex matrices. The method of this invention showed high and stable recovery rates for all twelve anthocyanins in the above six matrices, with the overall detection results exceeding the 70% pass rate. This indicates that the method of this invention can better meet the needs of simultaneous extraction and detection of anthocyanins with different matrix types and structures.

[0067] Therefore, compared with the method of Patent A, the method of the present invention can not only achieve simultaneous detection of twelve anthocyanins, but also has better overall performance in terms of applicability to complex matrices, uniform recovery of anthocyanins of different glycoside types, peak shape stability and quantitative reliability.

[0068] Comparison of Comparative Example 6 with the method disclosed in Chinese Patent CN111825730B The pretreatment and detection method disclosed in Chinese Patent CN111825730B was adopted as Patent Method B, and its detection effect on twelve anthocyanins in different matrix samples was compared with that of the present invention. The present invention uses L-carnitine-lactobionic acid-water as a natural eutectic solvent for ultrasonic-assisted extraction of samples, and simultaneously determines the twelve anthocyanins using the HPLC-MS / MS conditions described in the present invention.

[0069] The results are as follows Figure 11 As shown. By Figure 11 It is evident that Patent B method exhibits certain detection effectiveness for some matrix samples and some anthocyanins, with most anthocyanins achieving recoveries exceeding 70%. However, its recovery rate is significantly influenced by matrix type and anthocyanin structure, resulting in insufficient overall stability. When 70% is used as the acceptable recovery rate threshold, Patent B method shows recoveries below or close to 70% in some complex matrices. For example, in fish oil matrices, the recoveries of anthocyanins such as Pg3G, Cy3,5G, Del3,5G, Mal3,5G, Pg3,5G, Pn3,5G, and Pt3,5G are relatively low; in jelly matrices, the recoveries of anthocyanins such as Cy3,5G, Del3,5G, and Pg3,5G are close to or below 70%. These results indicate that Patent B method lacks adaptability for the extraction and detection of some anthocyanins in complex matrices, especially in deep-processed or composite matrices such as fish oil and jelly, making it difficult to guarantee acceptable recoveries for all target anthocyanins.

[0070] Further analysis reveals that Patent B method is primarily applicable to the detection of specific samples or certain anthocyanin components, and its pretreatment system has limited compatibility with anthocyanins of varying glycoside numbers, polarities, and stability. For a system of twelve target analytes simultaneously covering 3-O-glucoside and 3,5-O-diglucoside, Patent B method is prone to issues such as low recoveries of some components, significant fluctuations in recovery rates across different matrices, and insufficient applicability to complex matrices.

[0071] In contrast, this invention uses L-carnitine-lactobionic acid-water as a natural eutectic solvent for anthocyanin extraction. This system possesses strong polarity, an acidic microenvironment, and multiple hydrogen bonding interactions, simultaneously addressing the solubility, release efficiency, and extraction stability of both 3-O-glucoside and 3,5-O-diglucoside anthocyanins. This invention exhibits high and stable recoveries of all twelve anthocyanins in the aforementioned six matrices, generally exceeding the 70% threshold, with even more pronounced advantages in complex matrices such as fish oil and jelly. These results demonstrate that, compared to method B, this invention offers better applicability to complex matrices, superior overall recovery performance, and the ability to simultaneously extract anthocyanins with different structures, making it more suitable for the simultaneous detection and quantitative analysis of twelve anthocyanins in samples such as fresh berries, juices, jams, jellies, and berry-derived health products.

[0072] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for the simultaneous extraction and determination of twelve anthocyanins based on an L-carnitine-lactobionic acid-water eutectic solvent, characterized in that, The method includes the following steps: (1) Sample extraction Weigh the sample, add L-carnitine-lactobionic acid-water natural eutectic solvent, mix well, and then perform vortexing and ultrasonic-assisted extraction; after extraction, centrifuge and take the supernatant; dilute the supernatant with methanol aqueous solution containing formic acid or acetonitrile aqueous solution containing formic acid, vortex mix well, centrifuge again, and filter the supernatant through a microporous membrane to obtain the sample extract to be tested. (2) Preparation of matrix standard solution Using the blank matrix extract obtained in the same way as in step (1), a series of standard solutions of 12 anthocyanin mixed standards were prepared. (3) Anthocyanin content determination The series of anthocyanin mixed standard solutions prepared in step (2) and the sample extract prepared in step (1) were detected by HPLC-MS / MS. A standard curve was established with the concentration of each target anthocyanin as the abscissa and the corresponding chromatographic peak area or peak area ratio as the ordinate. The content of the twelve anthocyanins in the sample was calculated based on the response value of each target substance in the sample extract.

2. The method according to claim 1, characterized in that, The twelve anthocyanins include delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin-3,5-O-diglucoside, cyanidin-3,5-O-diglucoside, petunidin-3,5-O-diglucoside, pelargonidin-3,5-O-diglucoside, paeoniflorin-3,5-O-diglucoside, and malvidin-3,5-O-diglucoside.

3. The method according to claim 1, characterized in that, The preparation of the L-carnitine-lactobionic acid-water natural eutectic solvent in step (1) is as follows: L-carnitine, lactobionic acid and water are mixed in proportion, heated and stirred until a transparent and homogeneous liquid is formed, and then cooled to obtain the L-carnitine-lactobionic acid-water natural eutectic solvent.

4. The method according to claim 1, characterized in that, In the L-carnitine-lactobic acid-water natural eutectic solvent, the molar ratio of L-carnitine to lactobionic acid is 1:(0.2~3.0).

5. The method according to claim 1, characterized in that, The water content in the L-carnitine-lactobionic acid-water natural eutectic solvent is 15~65 wt% of the total mass of the L-carnitine-lactobionic acid-water natural eutectic solvent.

6. The method according to claim 1, characterized in that, The mass-to-volume ratio of the sample in step (1) to the natural eutectic solvent L-carnitine-lactobionic acid-water is 1:(5~60), g:mL.

7. The method according to claim 1, characterized in that, The dilution solution used in step (1) is a methanol-water-formic acid system or an acetonitrile-water-formic acid system; wherein the volume fraction of methanol or acetonitrile is 30~70%; and the volume fraction of formic acid is 0.5~3.0%.

8. The method according to claim 1, characterized in that, The HPLC-MS / MS detection conditions in step (3) are as follows: column: ACQUITY-UPLC-BEH-C18 (100 mm × 2.1 mm, 1.7 μm), column temperature 35~45℃; flow rate: 0.3~1.5 mL / min; mobile phase: A is formic acid aqueous solution, B is acetonitrile; gradient elution, injection volume: 2~5 μL.

9. The method according to claim 1, characterized in that, The mass spectrometry conditions for the HPLC-MS / MS determination in step (3) are as follows: electrospray ionization source, positive ion detection mode, multiple reaction monitoring mode; curtain gas CUR is 20~30 psi, collision gas CAD is Medium, ion spray voltage IS is 5400~5600 V, ion source temperature TEM is 450~550℃, ion source gas 1 GS1 is 55~65 psi, and ion source gas 2 GS2 is 35~45 psi.

10. The application of the method according to any one of claims 1 to 9 in the analysis of anthocyanin composition and quality evaluation of berries and their processed products.

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