A method for detecting the content of l-fuculose, trehalose and raffinose in a beverage

Abstract

The application provides a method for detecting the content of L-fucitol, trehalose and raffinose in a beverage. The method is optimized in the processes of sample extraction and purification, and thus the accuracy and reliability of the detection result are effectively improved, and the method can be applied to the detection of different types of beverages.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting the content of L-fucoitol, trehalose, and raffinose in beverages. Background Technology

[0002] L-fucoitol, trehalose, and raffinose are widely used in food, health products, cosmetics, and other fields, especially in food such as beverages.

[0003] Therefore, in order to ensure product consistency, meet labeling requirements, protect consumer health, and meet the needs of special groups, it is necessary to test the content of L-fucoitol, trehalose, and raffinose in beverages or other foods containing L-fucoitol, trehalose, and raffinose.

[0004] Currently, the technical difficulties in detecting L-fucoitol, trehalose, and raffinose in beverages may mainly include the following aspects: (1) Complex composition: Beverages usually contain a variety of components, including sugars, acids, pigments, preservatives, etc., which may interfere with the detection of L-fucoitol, trehalose, and raffinose, increasing the difficulty and complexity of detection. (2) Difficulty in detection: Due to their structural characteristics, L-fucoitol, trehalose, and raffinose do not have characteristic absorption under ultraviolet light and fluorescence, and cannot be detected using common ultraviolet detectors or fluorescence detectors. (3) Low content: In some beverages, the content of L-fucoitol, trehalose, and raffinose may be very low, which requires the detection method to have extremely high sensitivity and accuracy in order to accurately detect these low-content components.

[0005] In view of this, an accurate, reliable and simple detection method has been developed for the detection of L-fucoitol, trehalose and raffinose, which is of great significance to the beverage industry and even the food industry. Summary of the Invention

[0006] To address the problems and shortcomings of existing technologies, this invention provides a method for detecting the content of L-fucoitol, trehalose, and raffinose in beverages. This method, through optimization of the sample extraction and purification process, effectively improves the accuracy and reliability of the detection results and is applicable to the detection of different types of beverages.

[0007] This invention provides a method for detecting the content of L-fucoitol, trehalose, and raffinose in beverages, comprising the following steps: S1. Extraction: First, dissolve the sample in water, centrifuge, and take the first supernatant. Then, add a first organic solvent to the first supernatant and mix thoroughly to obtain a mixed solution; the volume ratio of the first supernatant to the first organic solvent is 10-30:70-90; the first organic solvent includes acetonitrile; S2. Purification: Transfer the mixed solution into an activated amino solid-phase extraction column. After the liquid in the column is completely discharged, elute with a second organic solvent. Collect all the eluent, remove the solvent from the eluent, redissolve, filter, and collect the filtrate; the second organic solvent includes methanol; S3. Prepare a standard curve for L-fucoitol, trehalose, and raffinose; S4. Inject the filtrate into a high-performance liquid chromatograph for detection, and substitute the detection results into the standard curve to determine the content of L-fucoitol, trehalose, and raffinose in the sample.

[0008] As mentioned in the background section above, current methods for detecting L-fucoitol, trehalose, and raffinose suffer from problems such as component complexity, detection difficulties, and low content, resulting in poor accuracy and sensitivity for these sugars or sugar alcohols. Therefore, proposing further solutions to address one of these problems and improve the accuracy and sensitivity of the detection of these sugars or sugar alcohols is particularly important for the food testing industry.

[0009] Therefore, the method for detecting the content of L-fucoitol, trehalose, and raffinose provided by this invention includes a specific sample purification step. In particular, an amino solid-phase extraction column is selected for sample purification, which effectively removes impurities from the sample. This largely avoids interference from other impurities and prevents misjudgments or missed detections caused by peaks from structurally similar materials. Furthermore, effective impurity removal makes the peaks of L-fucoitol, trehalose, and raffinose more pronounced, thereby improving the accuracy and sensitivity of the detection results. In addition, the amino solid-phase extraction column effectively retains the target compounds L-fucoitol, trehalose, and raffinose within the packing material, preventing them from flowing out with the loading and eluent used to remove interfering substances. Most interfering impurities flow out with the solvent, achieving the separation of fat-soluble vitamins, nucleotides, and water-soluble vitamins. Moreover, the amino solid-phase extraction column is suitable for different types of beverages; even beverages with significant differences in composition can achieve good purification results using this column. Furthermore, this invention also used other solid-phase extraction columns for purification. It was found that when using other solid-phase extraction columns, the target substances L-fucoitol, trehalose, and raffinose could not be effectively retained in these solid-phase extraction column packings and would flow out with the loading solution and eluent, making it difficult to achieve the effect of removing interfering substances.

[0010] Therefore, selecting a suitable loading solution is crucial for purification efficiency, based on the properties of the amino solid-phase extraction column. In the detection method for L-fucoitol, trehalose, and raffinose provided by this invention, firstly, in step S1, water is used as the solvent to extract the target analytes. Since these sugars or sugar alcohols are readily soluble in water, water extraction effectively extracts these substances, thus improving the accuracy and sensitivity of the final detection results. Further, acetonitrile is added for mixing; that is, acetonitrile and water are used together as the solvent for the target analytes. The final mixed solution obtained in step S1 (a mixture of the first supernatant and the first organic solvent) is used as the loading solution for purification using the solid-phase extraction column. It should be noted that because the contents of L-fucoitol, trehalose, and raffinose are relatively small, after water extraction, the solvent is essentially water, meaning that the main substance of the first supernatant is also water. Therefore, the second supernatant, as the loading solution for the solid-phase extraction column, has a volume ratio of water to acetonitrile of approximately 10–30:70–90, meaning that the volume percentage of acetonitrile is approximately 70–90%.

[0011] The reason for choosing a water-acetonitrile volume ratio of approximately 10–30:70–90 (i.e., the volume ratio of the first supernatant to acetonitrile) as the loading solution for the amino solid-phase extraction column is that the amino solid-phase extraction column works by retaining the target analyte through hydrogen bonding and dipole-dipole interactions of the amino functional groups. Hydrogen bonding weakens significantly with increasing water content, making it difficult to retain the target analyte in the solid-phase extraction column. Therefore, a high proportion of aqueous phase is unsuitable as a loading solution. Acetonitrile, due to its stable chemical structure, does not form hydrogen bonds with amino groups, thus not affecting the retention of the target analyte, making it suitable as a loading solution. However, since the target analyte is readily soluble in water but insoluble in acetonitrile, using pure acetonitrile as the loading solution would prevent the target analyte from dissolving in the solvent. Therefore, this invention selects acetonitrile + water as the loading solution. However, an excessively high proportion of aqueous phase weakens the retention capacity of the amino solid-phase extraction column, while an excessively low proportion would prevent the target analyte from completely dissolving. Therefore, the present invention selects a volume ratio of water to acetonitrile of approximately 10–30:70–90 as the loading solution (containing the target analyte, the content of which is low and can be temporarily ignored), which can take into account both the solubility of the target analyte and the removal of impurities, and is more conducive to the accuracy and sensitivity of the detection results.

[0012] After the sample is purified by an amino solid-phase extraction column, the target analyte remains inside the column. Therefore, it needs to be eluted to remove the analyte. The eluent should have sufficient polarity and solubility to ensure complete elution of the target substance from the solid-phase extraction column. This invention uses methanol as the eluent, which ensures high elution rates for L-fucoitol, trehalose, and raffinose, thus further guaranteeing higher accuracy and sensitivity of subsequent detection results.

[0013] It should be noted that in the testing industry, appropriate samples are typically taken and subjected to certain extraction steps. Because the sample size varies in different steps, the final analyte content calculated using the standard curve does not correspond to the analyte content of the initial sample. However, the analyte content of the initial sample can be calculated based on the changes in sample size in different steps. This invention follows the same principle; the conversion process will not be elaborated upon here, as it is a standard data processing operation in this field and will be understood by those skilled in the art.

[0014] Preferably, in step S1, the centrifugation speed is 3500–4500 r / min, and the centrifugation time is 2–5 min. Controlling the centrifugation speed is beneficial for fully extracting the target substance, achieving good separation between the target substance and impurities, and preventing damage to the target substance.

[0015] Preferably, in S1, the volume ratio of the first supernatant to the first organic solvent is 20:80.

[0016] Preferably, in S1, after adding the first organic solvent, the mixture is vortexed and homogenized, and then sonicated for 8–15 minutes.

[0017] Preferably, in step S2, the amino solid-phase extraction column is activated sequentially using a third organic solvent and water, wherein the third organic solvent includes methanol. Activating the amino solid-phase extraction column sequentially with methanol and water effectively removes impurities from the column and adjusts its polarity, making the extraction column suitable for the target analyte type in this invention. This provides greater flexibility in practical applications, allowing the ratio of methanol to water to be adjusted according to the specific analyte and sample matrix.

[0018] Preferably, in step S2, the amino solid-phase extraction column is activated sequentially with methanol and water, and the volume ratio of methanol to water is 8–20:8–20. Furthermore, activating the amino solid-phase extraction column with methanol and water in the above ratio is more suitable for the analysis of several sugars or sugar alcohols in this invention. This is more conducive to immobilizing the target analyte within the column while removing other impurities, thus facilitating subsequent detection and analysis and improving the accuracy and sensitivity of the detection results.

[0019] Preferably, in step S2, after transferring the mixed solution into the activated amino solid-phase extraction column, a mixed solvent, comprising water and acetonitrile, is added at a liquid level of 1.5–2.5 mm on the column. The volume ratio of water to acetonitrile is 10–30:70–90. Furthermore, after all the mixed solution obtained in step S1 has been transferred into the amino solid-phase extraction column, a certain amount of the water and acetonitrile mixed solvent is added. This facilitates more thorough separation of the target compound from impurities, achieving better impurity removal and further improving the accuracy and sensitivity of subsequent detection results.

[0020] Preferably, in the mixed solvent of S2, the volume ratio of water to acetonitrile is 20:80.

[0021] Preferably, in S2, the amino solid-phase extraction column comprises CNWBOND NH2 / Si SPE.

[0022] Preferably, the column specification of CNWBOND NH2 / Si SPE is 1g / 10mL.

[0023] Preferably, in step S2, the solvent in the eluent is removed using an inert gas at a temperature of 45–55°C. Preferably, in step S2, the solvent in the eluent is removed using an inert gas at a temperature of 50°C. Preferably, the inert gas includes at least one of nitrogen and argon.

[0024] Preferably, in step S2, the reconstitution solution used in the resolution process includes methanol and water; and the volume ratio of methanol to water is 80-92:8-20. Different solvents will produce solvent peaks due to different absorbance values ​​during sample injection, and different solvents have inconsistent solubility for the target analyte. Therefore, selecting a suitable reconstitution solution is beneficial for balancing the solubility of the target analyte and the peak quality, thereby ensuring higher accuracy and sensitivity of the detection results. Furthermore, this invention also uses other reconstitution solvents such as acetonitrile + water. However, during the experiment, it was found that excessive sugar content would cause acetonitrile + water to separate into layers. Therefore, if acetonitrile + water is chosen as the reconstitution solution, this experimental method is only suitable for detecting samples with relatively low sugar content. Using methanol + water as the reconstitution solvent allows for a wider detection range, effectively dissolving the sample, and the spectra do not show peak broadening or peak bifurcation, making it a better reconstitution solution.

[0025] Preferably, in S2, the resolution solution used in the resolution process includes methanol and water.

[0026] Preferably, in S2, the resolution process uses a resolution solution comprising methanol and water, wherein the volume ratio of methanol to water is 86:14.

[0027] Preferably, in S2, a 0.22μm microporous membrane is used for filtration.

[0028] Preferably, in S3, the correlation coefficients of the logarithmic regression equations for the standard curves of L-fucoitol, trehalose, and raffinose are all not less than 0.99.

[0029] Preferably, the standard curve for L-fucoitol is y = 2.0070.2538x. 1.34731475 Preferably, the standard curve for L-fucoitol is y = y = 2.0070.2538x. 1.34731475 The logarithmic working curve ranges from ρ to 0.04 to 0.8 mg / mL. -1 The correlation coefficient was 0.99929.

[0030] Preferably, the standard curve for trehalose is y = 7589.02174x. 1.28211932 Preferably, the standard curve for trehalose is y = 7589.02174x. 1.28211932 The linear range is 0.25–0.5 mg / mL. -1 The correlation coefficient is 0.99966.

[0031] Preferably, the standard curve for raffinose is y = y = 23436.582x 1.12890623 Preferably, the standard curve for raffinose is y = 23436.582x. 1.12890623 The linear range is 0.05–1.0 mg / mL. -1 The correlation coefficient is 0.99955.

[0032] Preferably, the specific steps for preparing standard curves for L-fucoitol, trehalose, and raffinose in step S3 are as follows:

[0033] Step 1: Prepare standard mixed working solutions for L-fucoitol, trehalose, and raffinose. The concentration points for L-fucoitol are 0.04 mg / mL, 0.08 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL; the concentration points for trehalose are 0.025 mg / mL, 0.05 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL; and the concentration points for raffinose are 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL.

[0034] Step 2: Plot a standard curve with the measured peak area of ​​the target analyte as the ordinate and the corresponding concentration of the standard mixed working solution as the abscissa. Preferably, the standard curve is a logarithmic standard working curve.

[0035] Preferably, in S4, the chromatographic column of the high-performance liquid chromatograph includes an amino column. Using this type of column, compared to other types, provides better peak shape and resolution, thus improving the accuracy and sensitivity of the detection results.

[0036] Preferably, the amino chromatography column has a size of 4.6 × 250 mm and the packing material (filler) has a particle size of 5 μm.

[0037] Preferably, in S4, the elution process of the high-performance liquid chromatograph is gradient elution, and the eluent includes mobile phase A and mobile phase B; mobile phase A is acetonitrile, mobile phase B is water, and the specific elution procedure is as follows:

[0038] 0.00 86 14 6.00 88 12 25.00 88 12 30.00 80 20 38.00 80 20 39.00 86 14 40.00 86 14

[0039] Using gradient elution and the above-mentioned eluent and specific elution procedure is more conducive to separating the target substances L-fucoitol, trehalose and raffinose, thus improving the accuracy and sensitivity of the detection results.

[0040] Preferably, in step S4, the injection volume of the high-performance liquid chromatograph is 15–30 μL. More preferably, in step S4, the injection volume of the high-performance liquid chromatograph is 20 μL.

[0041] Preferably, the column temperature in the high-performance liquid chromatograph is 43–47°C. More preferably, the column temperature in the high-performance liquid chromatograph is 45°C.

[0042] Preferably, the flow rate of the eluent in the high-performance liquid chromatograph is 0.8–1.2 mL / min. More preferably, the flow rate of the eluent in the high-performance liquid chromatograph is 1.0 mL / min.

[0043] Preferably, the evaporative light scattering detector (EVS) conditions in the high-performance liquid chromatograph (HPLC) are: nebulizer and drift tube temperature: 62–67°C; nitrogen flow rate: 1.4–1.8 L / min. Preferably, the EVS conditions in the HPLC are: nebulizer and drift tube temperature: 65°C; nitrogen flow rate: 1.6 L / min. Attached Figure Description

[0044] Figure 1 The chromatograms are of the eluents from different types of solid-phase extraction columns used in Example 1.

[0045] Figure 2 The chromatograms are of the eluents used in different types of loading solutions in Example 2.

[0046] Figure 3 The elution rate is shown in Example 3 after using different types of elution solutions.

[0047] Figures 4-6The image shows the resolution effect after using different types of resolution solutions in Example 4. Among them, Figure 4 This is a graph showing the redissolution results of 86% acetonitrile water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%. Figure 5 This is a graph showing the redissolution results of 86% acetone water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%. Figure 6 The graph shows the resolution results of 86% acetonitrile water with sugar contents of 0.5%, 1.0%, 1.5%, 2.0%, 5.0%, 10.0%, and 15.0%.

[0048] Figure 7 The chromatograms are those obtained using different types of chromatographic columns in Example 5; Figure 8 This is a chromatogram of the mobile phase used in Example 5.

[0049] Figure 9 The chromatogram of isocratic elution using 80% acetonitrile water in Example 5; Figure 10 This is a chromatogram of gradient elution used in Example 5. Detailed Implementation

[0050] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0051] The main instruments and apparatus used in the following examples are as follows: High-performance liquid chromatograph: Agilent Technologies 1260; Electronic balance: Sartorius BSA423S; Centrifuge: Xiangyi Laboratory Instrument Development Co., Ltd. L-550; Vortex mixer: IKA Vortex 1; Ultrasonic cleaner: Kunshan Ultrasonic Instrument Co., Ltd. KQ-5200DE; Nitrogen blower concentrator: Biotage Turbo Vap LV. The main reagents and materials used are as follows: Standard substances: L-fucoitol standard (C6H14O5, CAS No.: 13074-06-1): purity ≥98%; Trehalose standard (C12H22O11, CAS No.: 99-20-7): purity 99.8%; Raffinose (C18H32O16, CAS No.: 512-69-6): purity 94.0%. Reagents: Water (grade I), acetonitrile (chromatographic grade), methanol (chromatographic grade), acetone (chromatographic grade).

[0052] Example 1

[0053] Selection of solid phase extraction column:

[0054] In this embodiment, HLB, C18, WAX, and NH2 / Si solid-phase extraction columns were used for detection, respectively, and the following steps were followed:

[0055] Step 1: Add L-fucoitol, trehalose, and raffinose standards to 80% acetonitrile water (acetonitrile to water volume ratio of 80:20) to obtain a mixed standard solution.

[0056] Step 2: After activating the solid-phase extraction column (different solid-phase extraction columns are used according to the variables in this embodiment, namely HLB, C18, WAX, and NH2 / Si solid-phase extraction columns), the mixed standard solution from Step 1 is passed through the activated solid-phase extraction column. The solutions before and after entering the solid-phase extraction column are recorded as the loading solution and the eluent, respectively. The eluent is blown with nitrogen at 50°C until nearly dry, redissolved with 86% methanol aqueous solution, filtered through a 0.22μm microporous membrane, and the filtrate is collected in a sample vial for injection.

[0057] Step 3: Plot standard curves for L-fucoitol, trehalose, and raffinose respectively. For plotting standard curves for L-fucoitol, trehalose, and raffinose, refer to Example 6 of this invention (wherein, in the detection parameters of the high-performance liquid chromatograph, the elution program can refer to the gradient elution of Example 6, or the isocratic elution of this example, and the other parameters are consistent with this example). However, the specific standard curves may deviate slightly, and specific standard curves can be plotted according to the actual situation.

[0058] Step four: Analyze the eluent from step two using high-performance liquid chromatography (HPLC). Substitute the results into a standard curve to obtain the contents of L-fucoitol, trehalose, and raffinose in the eluent. The chromatogram of the eluent is shown below. Figure 1 As shown.

[0059] It should be noted that the HLB solid-phase extraction column (60mg / 3mL) was activated sequentially with 10mL methanol and 10mL water, the C18 solid-phase extraction column (500mg / 3mL) was activated sequentially with 10mL methanol and 10mL water, the WAX ​​solid-phase extraction column (500mg / 6mL) was activated sequentially with 10mL methanol and 10mL water, and the NH2 / Si solid-phase extraction column (1g / 10mL) was activated sequentially with 10mL methanol and 10mL water (methanol and water volume ratio of 1:1).

[0060] The detection conditions for high performance liquid chromatography are as follows:

[0061] Chromatographic column: Amino chromatography column (4.6×250mm, packing material (filler) particle size 5μm, diol-bonded silica gel as packing material);

[0062] Injection volume: 20 μL;

[0063] Column temperature: 45℃;

[0064] Flow rate: 1.0 mL / min;

[0065] Evaporative light scattering detector conditions: nebulizer and drift tube temperature: 65℃; nitrogen flow rate: 1.6 L / min;

[0066] Elution procedure: Mobile phase: Acetonitrile: Water (volume ratio) = 84:16 (The pretreatment optimization process uses standard substances, does not involve the sample matrix, and does not have high separation requirements. Therefore, isocratic elution is used to enable rapid detection of the target analyte and save optimization time).

[0067] Furthermore, in step four above, the contents of L-fucoitol, trehalose, and raffinose in the effluent are shown in Table 1, and the recovery rates calculated from the effluent are also shown in Table 1. Taking L-fucoitol as an example, the L-fucoitol recovery rate = L-fucoitol content in the effluent / L-fucoitol content in the loading solution, where the loading solution is the prepared mixed standard solution. The calculation method for the other two target compounds is similar. A higher recovery rate indicates a lower ability of the solid-phase extraction column to retain the three target compounds.

[0068] Table 1 shows the content and recovery rate of L-fucoitol, trehalose, and raffinose in the sample solution and effluent of step four in this embodiment.

[0069]

[0070] Depend on Figure 1 The results shown in Table 1 indicate that the recovery rates of the three target compounds (L-fucoitol, trehalose, and raffinose) in the effluent from the HLB, C18, and WAX solid-phase extraction columns were approximately 100%, meaning that all three target compounds eluted with the solvent. The corresponding packing material in the solid-phase extraction columns was insufficient to retain the target compounds, thus failing to achieve the purification mechanism designed in the experiment. However, no target compounds were detected in the effluent from the NH2 / Si SPE solid-phase extraction column, indicating that the three target compounds were effectively retained in the packing material of this solid-phase extraction column and did not elute with the solvent, which may contain impurities such as fat-soluble vitamins, nucleotides, and water-soluble vitamins, achieving a better purification effect. Therefore, this invention selects the NH2 / Si solid-phase extraction column, specifically the NH2 / Si SPE column, as the appropriate solid-phase extraction column, which is more conducive to the separation of the three target compounds and thus improves the accuracy and sensitivity of the detection results.

[0071] Example 2

[0072] Selection of sample loading solvent:

[0073] The working principle of the NH2 / Si solid-phase extraction column is that amino functional groups retain target analytes through hydrogen bonding and dipole-dipole interactions. Currently, the most commonly used solvents in the laboratory are methanol, water, and acetonitrile. Analysis of the chemical properties of these solvents reveals that methanol forms hydrogen bonds with the amino groups on the column surface, reducing the column's retention capacity for the target analytes; therefore, methanol is unsuitable as a loading solvent. Hydrogen bonding weakens significantly with increasing water content, making it difficult to retain the target analytes in the solid-phase extraction column; therefore, a high proportion of aqueous phase is unsuitable as a loading solvent. Acetonitrile, due to its stable chemical structure, does not form hydrogen bonds with amino groups, thus not affecting the retention of the target analytes; therefore, acetonitrile is suitable as a loading solvent. However, since the target analytes are readily soluble in water but insoluble in acetonitrile, using pure acetonitrile as the loading solvent will prevent the target analytes from dissolving in the solvent. Considering all these factors, this experiment selected acetonitrile-water as the loading solvent. However, an excessively high proportion of aqueous phase will weaken the retention capacity of the NH2 / Si solid-phase extraction column, while an excessively low proportion will prevent the target analytes from completely dissolving. Therefore, finding the right ratio of acetonitrile to water is crucial for the experiment.

[0074] Therefore, this embodiment uses different volume ratios of acetonitrile + water as the sample loading solvent, and the specific steps are as follows:

[0075] Step 1: L-fucoitol, trehalose, and raffinose standards were added to acetonitrile aqueous solutions with different volume ratios to obtain mixed standard solutions with different solvents. Here, based on actual variables, the volume ratio of acetonitrile to water was set to 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, and 85:15, respectively.

[0076] Step 2: After activating the solid-phase extraction column (using an NH2 / Si solid-phase extraction column, activated sequentially with 10 mL of methanol and 10 mL of water (methanol to water volume ratio of 1:1), the mixed standard solutions of different solvents from Step 1 are passed through the activated solid-phase extraction column. The solutions before and after entering the solid-phase extraction column are recorded as the loading solution and the eluent. The eluent is purged with nitrogen at 50°C until nearly dry, reconstituted with an 86% methanol aqueous solution (methanol to water volume ratio of 86:14), filtered through a 0.22 μm microporous membrane, and the filtrate is collected in a sample vial for injection.

[0077] Step 3: Plot standard curves for L-fucoitol, trehalose, and raffinose respectively. For plotting standard curves for L-fucoitol, trehalose, and raffinose, refer to Example 6 of this invention (wherein, in the detection parameters of the high-performance liquid chromatograph, the elution program can refer to the gradient elution of Example 6, or the isocratic elution of Example 1, and the other parameters are consistent with Example 1). However, the specific standard curves may deviate from the actual situation, and specific standard curves can be plotted according to the actual circumstances.

[0078] Step four: The eluent from step two was analyzed using high-performance liquid chromatography (HPLC). The results were then substituted into a standard curve to obtain the contents of L-fucoitol, trehalose, and raffinose in the eluent. The HPLC parameters were the same as in Example 1.

[0079] In the above steps, the content of the target analyte in the effluent from step two can be detected, and the content of the target analyte retained in the NH2 / Si solid-phase extraction column can be calculated accordingly. This allows for the calculation of the retention capacity of different loading solutions. (Target analyte content in the added standard - Target analyte content in the effluent) / Content of the added standard * 100% = Retention capacity. The target analyte content in the added standard is the same as the target analyte content in the loading solution. Only when the retention capacity reaches 100% can the target analyte be completely adsorbed onto the solid-phase extraction column, achieving the effect of separating the target analyte from impurities in the effluent.

[0080] After the above steps of testing, the results are as follows: Figure 2 As shown, Figure 2 The results showed that when 50% acetonitrile-water (representing a volume ratio of acetonitrile to water of 50:50) was used as the loading solvent, the retention rates of the three target compounds were all below 40%. Further increasing the acetonitrile-water ratio positively correlated the retention rates of the three target compounds. When 75% acetonitrile-water was used as the loading solvent, the retention rates of trehalose and raffinose reached the expected levels. However, at this ratio, the retention rate of L-fucoitol was only 75%, which was not conducive to the simultaneous processing of the three target compounds. Further increasing the acetonitrile-water ratio did not decrease the retention rates of trehalose and raffinose, while the retention rate of L-fucoitol also reached the optimal level. To balance the solubility of the target compounds in the loading solvent, 80% acetonitrile-water was the best loading solvent.

[0081] Example 3

[0082] Selection of elution solution:

[0083] The elution solvent should have sufficient polarity and solubility to ensure that the target substance can be completely eluted from the solid-phase extraction column. Therefore, this embodiment preferably uses ammoniated methanol, acetone, or methanol, which can react with amino groups in the solid-phase extraction column, for the elution experiment. The specific steps are as follows:

[0084] Step 1: Add L-fucoitol, trehalose, and raffinose standards to 80% acetonitrile water (acetonitrile to water volume ratio of 80:20) to obtain a mixed standard solution.

[0085] Step 2: After activating the solid-phase extraction column (an NH2 / Si solid-phase extraction column was used, and 10 mL of methanol and 10 mL of water (methanol and water volume ratio of 1:1) were used to activate the NH2 / Si solid-phase extraction column), the mixed standard solution from Step 1 was passed through the activated solid-phase extraction column until all liquid was discharged from the column. Elution was then performed using eluent, and all eluent was collected. The eluent was then purged with nitrogen at 50°C until nearly dry, and reconstituted using an 86% methanol-water solution (methanol and water volume ratio of 86:14). The solution was then filtered through a 0.22 μm microporous membrane, and the filtrate was collected in a sample vial for injection. In this step, depending on the variables in this embodiment, different solvents (of the same volume) were used as eluents for the target analyte elution. These different solvents (different eluents) included ammoniated methanol, acetone, and methanol.

[0086] Step 3: Plot standard curves for L-fucoitol, trehalose, and raffinose respectively. For plotting standard curves for L-fucoitol, trehalose, and raffinose, refer to Example 6 of this invention (wherein, in the detection parameters of the high-performance liquid chromatograph, the elution program can refer to the gradient elution of Example 6, or the isocratic elution of Example 1, and the other parameters are consistent with Example 1). However, the specific standard curves may deviate from the actual situation, and specific standard curves can be plotted according to the actual circumstances.

[0087] Step four: The filtrate from step two is analyzed using high-performance liquid chromatography (HPLC). The results are then substituted into a standard curve to obtain the contents of L-fucoitol, trehalose, and raffinose in the eluent. The HPLC parameters are the same as in Example 1.

[0088] In the above steps, by detecting the content of the target analyte in the eluent, the content of the target analyte retained in the eluent can be calculated, and thus the elution capacity of different eluents can be calculated. (Target analyte content in eluent) / (Added standard content) * 100% = Elution capacity (elution rate), where the added standard content is the same as that in step one. Only when the elution capacity is close to 100% can almost all of the target analyte elute from the solid-phase extraction column, meeting the detection requirements.

[0089] After the above steps of testing and related calculations, the results are as follows: Figure 3 As shown, Figure 3The results showed that ammoniated methanol could elute 54.6% of L-fucoitol and 79.4% of raffinose, while the elution rate of trehalose was only 12.2%; acetone could elute 99.8% of L-fucoitol and 69.9% of trehalose, while the elution rate of raffinose was essentially 0; methanol could elute 98.9% of L-fucoitol, 99.6% of trehalose, and 99.4% of raffinose. Therefore, methanol exhibited the highest elution rate for all three target compounds, demonstrating excellent and balanced elution performance for all three.

[0090] Example 4

[0091] Selection of reconstitution solution:

[0092] This embodiment uses different types of reconstitution solutions as variables to explore the reconstitution effect of different types of reconstitution solutions. The specific steps are as follows:

[0093] Using a 4g sample with a sugar content of 0.5% (mass fraction) (sugar content 4g * 0.5% = 0.02g), dilute the 4g sample to 10ml with water. Then, take 1.5ml and pass it through a solid-phase extraction column (NH2 / Si solid-phase extraction column, which is activated sequentially with 10mL methanol and 10mL water). After the liquid in the column is completely discharged, elute with methanol and collect all the eluent. Blow the eluent to near dryness with nitrogen at 50℃, reconstitute with a reconstitution solution, and dilute to 1mL. Filter through a 0.22μm microporous membrane and collect the filtrate. The sugar content in the solid-phase extraction column should be 0.02g * 1.5ml / 10ml = 0.003g. When reconstituted with a 0.5% sugar content sample, 1ml of reconstitution solution needs to reconstitute 0.003g of sugar.

[0094] To facilitate more direct observation of the process, this experiment used 10 ml of a reconstitution solution to reconstitute a sample containing 0.03 g of sugar to simulate the pretreatment process of reconstituted a 0.5% sugar content sample using 1 ml of a reconstitution solution. 0.25 g of sucrose was weighed, diluted to 10 ml with water, mixed well, and then 1.2 ml was transferred to make the sugar content in the aqueous phase 0.03 g. 8.8 mL of acetonitrile, acetone, and methanol were added respectively to simulate the reconstitution of a 5% sugar content sample between a 14% aqueous phase and an 86% organic phase. If layering or precipitation was observed, it indicated that the reconstitution effect did not meet the experimental requirements and subsequent testing was not possible. This process was repeated for reconstitution observations of sugar contents of 1.0%, 1.5%, 2.0%, 5.0%, 10.0%, and 15.0%.

[0095] The results of its redissolution are as follows Figure 4 , 5 As shown in Figure 6, Figure 4 This is a graph showing the redissolution results of 86% acetonitrile water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%. Figure 5This is a graph showing the redissolution results of 86% acetone water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%. Figure 6 This is a graph showing the reconstitution results of 86% acetonitrile water with sugar contents of 0.5%, 1.0%, 1.5%, 2.0%, 5.0%, 10.0%, and 15.0%. (Source: [Insert graph here]) Figure 4 It can be seen that when acetonitrile-water was used to reconstitute a sample simulating a 0.5% sugar content, obvious stratification occurred, and the amount of the lower, acetonitrile-poor phase increased with increasing sugar content. Therefore, acetonitrile-water is not suitable as a reconstitution solution. Subsequently, the laboratory attempted to reconstitute the sample using acetone-water. Figure 5 The results showed that samples with a sugar content of 0.5% showed good resolution with acetone-water, but significant solid precipitation occurred when the sugar content was 1.0%. The resolution ability was negatively correlated with increasing sugar content, indicating that acetone-water is only suitable for resolution of samples with low sugar content. To find a resolution solution with better resolution ability, the laboratory further selected methanol-water for testing (isopropanol and ethanol, among alcohols, have high viscosity, which would lead to a significant increase in column pressure, making them unsuitable as alternative resolution solutions). Through observation... Figure 6 The study found that even when reconstituted with methanol-water, samples with a sugar content as high as 15% did not undergo sugar precipitation or stratification, indicating that the reconstitution capacity of methanol-water fully met the experimental requirements. Therefore, methanol-water is an excellent choice for the reconstitution solution in this experiment.

[0096] Example 5

[0097] (1) Optimization of computer lab conditions:

[0098] Firstly, this embodiment uses different types of chromatographic columns as variables to explore the detection effects of different types of chromatographic columns. The specific steps are as follows:

[0099] Step 1: Using methanol-water (methanol to water volume ratio of 86:14) as solvent, prepare a standard mixed solution for use with L-fucoitol, trehalose, and raffinose (containing L-fucoitol at a concentration of 0.2 mg / mL, trehalose at a concentration of 0.125 mg / mL, and raffinose at a concentration of 0.25 mg / mL).

[0100] Step 2: Inject the standard mixed solution into the high-performance liquid chromatograph (HPLC) for detection. In this step, according to the variables in the first aspect of this embodiment, the different column types in the HPLC (including C18 column, glycol column, and amino column, all three columns have a diameter (25 cm), inner diameter (4.6 mm), and packing particle size (5 μm) for detection. The other detection conditions of the HPLC are the same as in Example 1.

[0101] Test results are as follows Figure 7As shown, the target analytes on the C18 column were all detected within 2.5 minutes, indicating no retention or aggregation of the target analytes by this column. Both the diol-based and amino-based columns demonstrated good separation and retention of the three target analytes, meeting the detection requirements. A careful comparison of the two spectra revealed no significant difference in peak area between the amino and diol-based columns for L-fucoitol. However, the peak areas for trehalose and raffinose detected using the diol-based column were only about one-third of those detected using the amino-based column. A higher peak area for the same concentration of compound indicates higher instrument sensitivity, lower detection limits, improved accuracy, and increased data reliability and repeatability. Therefore, the amino-based column is the better choice for this experiment. Thus, selecting the amino-based column offers significant advantages and is more conducive to improving the overall accuracy and sensitivity of the detection results.

[0102] (2) Selection of mobile phase:

[0103] Secondly, this embodiment uses different mobile phases as variables to explore the detection effect of different mobile phases. The specific steps are as follows:

[0104] Step 1: Using methanol-water (methanol to water volume ratio of 86:14) as solvent, prepare a standard mixed solution for use with L-fucoitol, trehalose, and raffinose (containing L-fucoitol at a concentration of 0.2 mg / mL, trehalose at a concentration of 0.125 mg / mL, and raffinose at a concentration of 0.25 mg / mL).

[0105] Step 2: Inject the standard mixed solution into the high-performance liquid chromatograph (HPLC) for detection. In this step, according to the variables in the second aspect of this embodiment, the type of mobile phase is adjusted (including 86% methanol-water (methanol:water volume ratio of 86:14) and 80% acetonitrile-water (acetonitrile:water volume ratio of 86:14)). The remaining detection parameters of the HPLC are the same as in Example 1.

[0106] Test results are as follows Figure 8 As shown, using methanol-water as the mobile phase failed to effectively separate the three target analytes, while using acetonitrile-water as the mobile phase did. This is because methanol undergoes hydrogen bonding with the amino functional groups of the amino column, reducing the column's retention capacity for the target analytes. Consequently, the target analytes are detected within the first three minutes, failing to achieve effective separation. Acetonitrile, due to its chemical molecular structure, does not readily react with the packing material in the amino column. Furthermore, its low viscosity and low surface tension contribute to improved resolution and sensitivity in liquid chromatography, resulting in good performance during detection. This further promotes the effective separation of the three target analytes, thereby enhancing the overall accuracy and sensitivity of the detection results.

[0107] (3) Selection of mobile phase elution program:

[0108] Thirdly, this embodiment also optimized the mobile phase elution procedure. Since the target analyte is similar in properties to fructose, glucose, sucrose, maltose, and lactose commonly found in beverages, the laboratory optimization of the mobile phase elution procedure focused on whether it could effectively analyze the target analyte and fructose, glucose, sucrose, maltose, and lactose. First, the separation capability of isocratic elution with 80% acetonitrile-water was explored, and the results are as follows... Figure 9 As shown, Figure 9 It was found that the retention times of the characteristic peaks of trehalose overlapped with those of lactose and maltose (lactose and maltose overlapped), and the separation effect could not achieve the expected results.

[0109] Furthermore, the laboratory subsequently explored the use of gradient elution. During the validation process, it was found that the amino column is highly sensitive to changes in the mobile phase; excessively rapid changes in the mobile phase lead to large baseline fluctuations, making frequent and drastic mobile phase changes unsuitable. After repeated validation, the following elution programs were found to provide good separation results: 0.00–6.00 min (14%–12% B), 6.00–25.00 min (12% B), 25.00–30.00 min (12%–20% B), 30.00–38.00 min (20% B), 38.00–39.00 min (20%–14% B), and 39.00–40.00 min (14% B). All three target analytes were effectively separated from five common sugars found in experiments and beverages. The separation effect was as follows: Figure 10 As shown, the elution procedure meets the detection requirements. Therefore, using the above elution procedure is more conducive to improving the overall accuracy and sensitivity of the detection results.

[0110] Example 6

[0111] Limit of quantitation, standard curve, accuracy and precision:

[0112] The sample was tested according to the following steps:

[0113] S1. Extraction: First, dissolve the sample in water (weigh 4g of sample into a 10mL volumetric flask and dilute to the mark with water). After centrifugation, the centrifugation speed is 4000r / min and the centrifugation time is 3min. Take 1.5mL of the first supernatant into a 15mL centrifuge tube, then add 6mL of acetonitrile, vortex mix evenly, and sonicate for 10min. Collect the resulting mixed solution. In this embodiment, the volume ratio of the first supernatant to acetonitrile is recorded as 20:80 (because the sugar sample is mainly water with a very low sugar content, the first supernatant can be approximated as all water).

[0114] S2. Purification: Transfer the mixed solution obtained in S1 into an activated NH2 / Si solid-phase extraction column (activated sequentially with 10 mL methanol + 10 mL water (methanol:water volume ratio 1:1). The specifications of the NH2 / Si solid-phase extraction column are 1 g / 10 mL). Then, at a liquid level of 2.0 mm, add 2 mL of 80% acetonitrile-water solution (acetonitrile:water volume ratio 80:20). After the liquid in the column is completely discharged, elute with 25 mL methanol. Collect all eluent, blow it to near dryness with nitrogen at 50 °C, redissolve it with 86% methanol-water solution (methanol:water volume ratio 86:14), and make up to 1 mL. Filter through a 0.22 μm microporous membrane and collect the filtrate for injection.

[0115] S3. Construct standard curves for L-fucoitol, trehalose, and raffinose;

[0116] S4. Inject the filtrate into a high-performance liquid chromatograph (HPLC) for detection, and substitute the detection results into the standard curve to determine the content of L-fucoitol, trehalose, and raffinose in the sample. The detection conditions of the HPLC are the same as in Example 1, except for the elution program. The specific elution program is: gradient elution, with the eluent including mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is water. The specific elution program is shown in Table 2 below:

[0117] Table 2. Specific procedures for gradient elution

[0118] 0.00 86 14 6.00 88 12 25.00 88 12 30.00 80 20 38.00 80 20 39.00 86 14 40.00 86 14

[0119] The preparation steps for the standard curves of L-fucoitol, trehalose, and raffinose in S3 are as follows:

[0120] Standard stock solution (20 mg / mL): Weigh 0.5 g of L-fucoitol, trehalose and raffinose respectively (accurate to 0.0001 g), dissolve in 5 mL of water, and dilute to 25 mL with methanol.

[0121] Mixed standard working solution (L-fucoitol: 4 mg / mL, trehalose: 2.5 mg / mL, raffinose: 5 mg / mL): Accurately transfer appropriate amounts of L-fucoitol, trehalose, and raffinose standard stock solutions into a 25 mL volumetric flask, and dilute to the mark with methanol-water solution (volume ratio 86:14) to prepare a mixed standard working solution containing 4 mg / mL L-fucoitol, 2.5 mg / mL trehalose, and 5 mg / mL raffinose.

[0122] Standard mixed working solutions: Accurately pipette 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, and 400 μL respectively, and dilute to 1 mL with methanol-water solution (86:14, v / v). Prepare standard mixed working solutions (containing L-fucoitol at concentrations of 0.04 mg / mL, 0.08 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL; trehalose at concentrations of 0.025 mg / mL, 0.05 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL; and raffinose at concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL).

[0123] Plotting standard curves: The L-fucoitol, trehalose, and raffinose solutions of different concentrations were injected into a high-performance liquid chromatograph (HPLC) for testing (the HPLC testing conditions could be the same as in Example 1; alternatively, the isocratic elution of Example 1 could be replaced with gradient elution in this example, with the remaining detection conditions consistent with Example 1). Standard curves were plotted with the peak area of ​​the target analyte as the ordinate and the corresponding concentration of the standard mixed working solution as the abscissa. The relevant information for the obtained standard curves is shown in Table 3.

[0124] Table 3. Information related to the standard curve

[0125]

[0126] After plotting the relevant standard curve in step S3, the sample detection step in step S4 is performed. For negative samples (samples excluding the target analyte, also known as blank samples), a three-level, six-parallel spiked recovery experiment is conducted at the limit of quantitation (LOQ), twice the LOQ, and ten times the LOQ to verify accuracy and precision. The experimental results show that the recovery rate of L-fucoitol is 95.63–102.50%, with an RSD of 1.19–2.45%; the recovery rate of trehalose is 92.00–102.00%, with an RSD of 2.27–2.56%; and the recovery rate of raffinose is 96.00–103.10%, with an RSD of 2.17–2.90%. The accuracy and precision of the method meet the requirements of GB / T 27404-2008 standard. Specific values ​​are shown in Table 4. The above results demonstrate that the detection method provided by this invention has high accuracy and precision. For example, taking the limit of quantitation of the spiked level in Table 4 as 0.08 g / kg, 0.08 g / kg represents the spiked concentration, which is the detection state when 1 kg of sample contains 0.08 g of the target substance.

[0127] Table 4. Results of Accuracy and Precision Verification

[0128]

[0129] Example 7

[0130] Re-verification of different samples:

[0131] To verify the applicability of the detection method provided by this invention to different beverages, several commercially available beverages that commonly contain added sweeteners (carbonated beverages, vitamin functional beverages, plant protein beverages, and tea beverages) were selected for quantitation-limited level spiked recovery experiments to re-verify the accuracy parameters. The specific testing steps are as described in Example 6. The final verification results are shown in Table 5. The results indicate that the spiked recoveries of all matrices meet the requirements of GB / T 27404-2008 standard, demonstrating that the detection method provided by this invention is suitable for detecting different types of beverages and has a wide range of applications.

[0132] Table 5. Spike recovery results for different samples

[0133]

[0134]

[0135] In summary, this invention employs solid-phase extraction for pretreatment and high-performance liquid chromatography (HPLC) to determine the content of L-fucoitol, trehalose, and raffinose in beverages. The established detection method has been applied to different types of beverages with good results. This method is highly precise and accurate, time-efficient, and easy to operate, enabling the simultaneous detection of three sweeteners. It provides a new detection technique and basis for the simultaneous detection of multiple target compounds, including sugars and sugar alcohols with different chemical structures.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage, characterized in that, Includes the following steps: S1. Extraction: First, the sample is dissolved in water, centrifuged, and the first supernatant is taken. Then, a first organic solvent is added to the first supernatant and mixed evenly to obtain a mixed solution. The volume ratio of the first supernatant to the first organic solvent is 10-30:70-90. The first organic solvent includes acetonitrile. S2. Purification: The mixed solution is transferred into an activated amino solid-phase extraction column. Then, at a liquid level of 1.5–2.5 mm on the column, a mixed solvent is added until the liquid in the column is completely discharged. Elution is then performed using a second organic solvent. All eluent is collected, the solvent in the eluent is removed, the solution is reconstituted, filtered, and the filtrate is collected. The second organic solvent includes methanol. The mixed solvent includes water and acetonitrile, with a volume ratio of water to acetonitrile of 10–30:70–90. During the reconstitution process, the reconstituted solution includes methanol and water, with a volume ratio of methanol to water of 80–92:8–20. S3. Construct standard curves for L-fucoitol, trehalose, and raffinose; S4. The filtrate is injected into a high-performance liquid chromatograph (HPLC) for detection, and the detection result is substituted into the standard curve to determine the content of L-fucoitol, trehalose, and raffinose in the sample; the elution process of the HPLC is gradient elution, and the eluent includes mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is water, and the specific elution procedure is as follows: 。 2. The method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage as described in claim 1, characterized in that: In S1, during centrifugation, the centrifugation speed is 3500–4500 r / min, and the centrifugation time is 2–5 min.

3. The method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage as described in claim 1, characterized in that: In step S2, the amino solid-phase extraction column is activated sequentially using a third organic solvent and water, wherein the third organic solvent includes methanol.

4. The method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage as described in claim 1, characterized in that: In S2, the amino solid-phase extraction column comprises CNWBOND NH2 / Si SPE.

5. The method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage as described in claim 1, characterized in that: In S3, the R-values ​​of the linear regression equations for the standard curves of L-fucoitol, trehalose, and raffinose are respectively... 2 All values ​​are not lower than 0.

99.

6. The method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage as described in claim 5, characterized in that, The specific steps for generating standard curves for L-fucoitol, trehalose, and raffinose in step S3 are as follows: Step 1: Prepare standard mixed working solutions of L-fucoitol, trehalose, and raffinose, wherein the concentration points of L-fucoitol are 0.04 mg / mL, 0.08 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL; the concentration points of trehalose are 0.025 mg / mL, 0.05 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL; and the concentration points of raffinose are 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL. Step 2: Plot the standard curve with the measured peak area of ​​the target substance as the vertical axis and the corresponding concentration of the standard mixed working solution as the horizontal axis.

7. The method for detecting the content of L-fucoitol, trehalose, and raffinose in a beverage as described in claim 1, characterized in that, In S4, the chromatographic column of the high-performance liquid chromatograph includes an amino column.

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