Method for detecting contents of L-fucose, trehalose and raffinose in beverage

Abstract

According to the method for detecting the content of the L-fucose, the trehalose and the raffinose in the beverage, certain optimization is carried out in the sample extraction and purification process, the accuracy and reliability of the detection result are effectively improved, and the method can be suitable for detection of different types of beverages.

Description

Technical Field

[0001] The present invention belongs to the technical field of food detection, and particularly relates to a method for detecting the contents of L-fucitol, trehalose, and raffinose in beverages. Background Art

[0002] L-fucitol, trehalose, and raffinose are widely used in the fields of food, health products, cosmetics, etc., and are particularly widely used in foods such as beverages.

[0003] Therefore, in order to ensure product consistency, meet label identification requirements, protect consumer health, and meet the needs of special populations, it is necessary to detect the contents of L-fucitol, trehalose, and raffinose in beverages or other foods containing them.

[0004] Currently, the technical difficulties in detecting L-fucitol, trehalose, and raffinose in beverages may mainly include the following aspects: (1) Component complexity: Beverages usually contain various components, including sugars, acids, pigments, preservatives, etc. These components may interfere with the detection of L-fucitol, trehalose, and raffinose, increasing the difficulty and complexity of detection. (2) Detection difficulty: Due to their own structural characteristics, L-fucitol, 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 contents of L-fucitol, 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, developing an accurate, reliable, and simple detection method for the detection of L-fucitol, trehalose, and raffinose is of great significance for the beverage industry and even the food industry. Summary of the Invention

[0006] To solve the problems and deficiencies in the prior art, the present invention provides a method for detecting the contents of L-fucitol, trehalose, and raffinose in beverages. This method effectively improves the accuracy and reliability of the detection results by optimizing the sample extraction and purification processes, and can be applied to the detection of different types of beverages.

[0007] The present invention provides a method for detecting the contents of L-fucitol, trehalose, and raffinose in beverages, comprising the following steps: S1. Extraction: First, dissolve the sample with water, after centrifugation, take the first supernatant, and then add a first organic solvent to the first supernatant and mix 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: Transfer the mixed solution into an activated amino solid-phase extraction column. After the liquid in the column is completely drained, use a second organic solvent for elution. After collecting all the eluates, remove the solvent in the eluate, redissolve, filter, and collect the filtrate; the second organic solvent includes methanol; S3. Prepare a standard curve for L-fucitol, 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 contents of L-fucitol, trehalose, and raffinose in the sample.

[0008] As mentioned in the above background art, in the current detection methods for the contents of L-fucitol, trehalose, and raffinose, there are problems such as component complexity, difficult detection, and low content, resulting in poor detection accuracy and sensitivity for these sugars or sugar alcohols. Therefore, how to further propose a solution to one of the above problems to improve the detection accuracy and sensitivity of the above sugars or sugar alcohols is particularly important for the food detection industry.

[0009] Therefore, in the method for detecting the contents of L-fucitol, trehalose, and raffinose provided by the present invention, a treatment step for purifying the sample is specifically set. In particular, an amino solid-phase extraction column is selected to purify the sample, which can play a good role in removing impurities in the sample. Therefore, to a great extent, it avoids the interference of other impurity components on the detection, and also avoids the situation of misjudgment or missed judgment during the detection due to the elution of materials with similar structures. Moreover, after effectively removing the impurities, the elution peaks of L-fucitol, trehalose, and raffinose become more obvious, thereby improving the accuracy and sensitivity of the detection results. In addition, using the amino solid-phase extraction column can well retain the target substances L-fucitol, trehalose, and raffinose in the packing material and will not flow out with the sample loading solution and eluent for removing interfering substances, while most of the interfering impurity substances flow out with the solvent, achieving the effect of separating impurities such as fat-soluble vitamins, nucleotides, and water-soluble vitamins. And the amino solid-phase extraction column is applicable to different types of beverages, and even beverages with large compositional differences can achieve good purification effects after using the amino solid-phase extraction column. Also, the present invention also uses other solid-phase extraction columns for purification treatment and finds that when using other solid-phase extraction columns, the target substances L-fucitol, trehalose, and raffinose cannot be effectively retained in the packing materials of these solid-phase extraction columns and will flow out with the sample loading solution and eluent, making it difficult to achieve the effect of removing interfering substances.

[0010] Therefore, according to the properties of the amino solid-phase extraction column, it is also particularly important to select a suitable sample loading solution for the purification effect. In the method for detecting the contents of L-fucitol, trehalose, and raffinose provided by the present invention, first, in S1, water is used as a solvent to extract the target substances. Since these sugars or sugar alcohols can be fully dissolved in water, water extraction can fully extract these substances, which is beneficial to improving the accuracy and sensitivity of the final detection results. Further, acetonitrile is added and mixed, that is, acetonitrile and water are used together as the solvent for the target substances. That is, the mixed solution obtained finally in S1 (the first supernatant and the first organic solvent are mixed) is used as the sample loading solution for purification using the solid-phase extraction column. It should be noted here that because the contents of L-fucitol, trehalose, and raffinose are relatively small, after extraction with water, the solvent is basically water, that is, the main substance of the first supernatant is also water, which can be approximately regarded as the first supernatant being all water. Therefore, for the second supernatant as the sample loading solution for the solid-phase extraction column, the volume ratio of the solvent water to acetonitrile is equivalent to 10 - 30:70 - 90, that is, the volume ratio of acetonitrile is approximately 70 - 90%.

[0011] And why is the volume ratio of solvent water to acetonitrile about 10 - 30:70 - 90 (i.e., the volume ratio of the first supernatant to acetonitrile) selected as the sample loading solution for the amino solid-phase extraction column? This is because the working principle of the amino solid-phase extraction column is that the amino functional group retains the target substances through hydrogen bonding and dipole-dipole interactions. The hydrogen bonding will be significantly weakened as the proportion of water increases, making it difficult to retain the target substances in the solid-phase extraction column. Therefore, a high proportion of the water phase is not suitable as the sample loading solution; acetonitrile, due to its stable chemical structure, will not form hydrogen bonds with the amino group, thereby affecting the retention of the target substances. So acetonitrile is suitable as the sample loading solution. However, since the target substances are easily soluble in water and insoluble in acetonitrile, if pure acetonitrile is selected as the sample loading solution, the target substances cannot be dissolved in the solvent. Considering the above, the present invention selects acetonitrile + water as the sample loading solution. However, if the proportion of the water phase is too high, the retention ability of the amino solid-phase extraction column will be weakened; if the proportion of the water phase is too low, the target substances cannot be completely dissolved. Therefore, the present invention selects the volume ratio of solvent water to acetonitrile about 10 - 30:70 - 90 as the sample loading solution (including the target substances, the content of the target substances is low and can be temporarily ignored), which can balance the solubility of the target substances and the function of removing impurities, and is more beneficial to the accuracy and sensitivity of the detection results.

[0012] After the sample is purified by an amino solid-phase extraction column, the target substance will remain in the extraction column. Therefore, it is necessary to elute it to wash down the target substance. 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. In the present invention, methanol is used as the elution solvent, which can ensure a high elution rate of L-fucitol, trehalose, and raffinose, and thus can further ensure higher accuracy and sensitivity of the subsequent detection results.

[0013] Here, it should be noted that in the testing industry, usually an appropriate sample is taken through certain extraction steps. Since the sampling amount varies in different steps, the content of the analyte finally calculated by substituting into the standard curve is not the content of the analyte corresponding to the original sample. However, the content of the analyte in the original sample can be converted according to the change in the sampling amount in different steps. The same is true for the present invention, and the conversion process will not be elaborated here one by one. This is a routine operation in data processing in this field, and those skilled in the art can also understand it.

[0014] Preferably, in S1, during the centrifugation process, the centrifugation speed is 3500 - 4500 r / min, and the centrifugation time is 2 - 5 min. Controlling a certain rotation speed during centrifugation is beneficial to fully extract the target substance, simultaneously achieving good separation of the target substance from impurities, and also 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, it is mixed evenly by vortexing and ultrasonicated for 8 - 15 min.

[0017] Preferably, in S2, the amino solid-phase extraction column is activated successively with a third organic solvent and water. The third organic solvent includes methanol. Selecting methanol and water successively to activate the amino solid-phase extraction column can not only effectively remove impurities in the column but also adjust the polarity of the column, making the extraction column suitable for the type of target substances in the present invention. In practical applications, it is more flexible and the ratio of methanol to water can be adjusted according to specific analytes and sample matrices.

[0018] Preferably, in S2, the amino solid-phase extraction column is activated successively with methanol and water, and the volume ratio of methanol to water is 8 - 20:8 - 20. Further, 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 the present invention, that is, it is more conducive to fixing the target substances in the column while removing other impurities, and thus is more conducive to further detection and analysis later, improving the accuracy and sensitivity of the detection results.

[0019] Preferably, in S2, after transferring the mixed solution into the activated amino solid-phase extraction column, a mixed solvent needs to be added at a position where the liquid level on the column is 1.5 - 2.5 mm. The mixed solvent includes water and acetonitrile, and the volume ratio of water to acetonitrile is 10 - 30:70 - 90. Further, after all the mixed solution obtained in S1 is transferred into the amino solid-phase extraction column, a certain amount of water-acetonitrile mixed solvent is continuously added, which is beneficial to more fully separate the target compound from impurities, achieving a better impurity removal effect, 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 includes CNWBOND NH 2 / Si SPE.

[0022] Preferably, CNWBOND NH 2 The column specification of / Si SPE is 1 g / 10 mL.

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

[0024] Preferably, in S2, during the reconstitution process, the reconstitution solution used includes methanol and water; and the volume ratio of methanol to water is 80 - 92:8 - 20. When different solvents are injected, solvent peaks will appear due to different absorption values, and different solvents also have inconsistent solubilities for the target substance. Therefore, choosing a suitable reconstitution solution is beneficial to taking into account the solubility of the target substance and the peak quality, and further beneficial to ensuring higher accuracy and sensitivity of the detection results. Moreover, the present invention also uses other reconstitution solvents such as acetonitrile + water, but it is found during the experiment that excessive sugar content will cause acetonitrile and water to separate. Therefore, if acetonitrile-water is selected 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 can have a wider range of detections, can effectively dissolve the sample, and no peak broadening or peak splitting phenomenon appears in the chromatogram during injection, which is a better reconstitution solution.

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

[0026] Preferably, in S2, during the reconstitution process, the reconstitution solution used includes methanol and water, and the volume ratio of methanol to water is 86:14.

[0027] Preferably, in S2, filtration is carried out using a 0.22 μm microporous filter membrane.

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

[0029] Preferably, the standard curve for L-fucitol is y = 2.007 + 0.2538x 1.34731475 Preferably, the standard curve for L-fucitol is y = y = 2.007 + 0.2538x 1.34731475 , and the logarithmic working curve range ρ is 0.04 - 0.8 mg / mL -1 , and the correlation coefficient is 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 , and the linear range is 0.25 - 0.5 mg / mL -1 , and 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 , and the linear range is 0.05 - 1.0 mg / mL -1 , and the correlation coefficient is 0.99955.

[0032] Preferably, the specific operations for making the standard curves of L-fucitol, trehalose, and raffinose in S3 are as follows:

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

[0034] Step 2, with the measured peak area of the target as the ordinate and the concentration of the corresponding standard mixed working solution as the abscissa, plot the standard curve. 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 chromatographic column. Selecting this type of chromatographic column can provide better peak shape and resolution compared to other types of chromatographic columns, and thus is more conducive to improving the accuracy and sensitivity of the detection results.

[0036] Preferably, the size of the amino chromatographic column is 4.6×250 mm, and the particle size of the packing (filler) is 5 μm.

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

[0038] Time / min A / ％ B / ％ 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] Selecting gradient elution, the above-mentioned eluent, and the specific elution program is more conducive to separating the target substances L-fucitol, trehalose, and raffinose, and thus is beneficial to improving the accuracy and sensitivity of the detection results.

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

[0041] Preferably, the column temperature of the chromatographic column in the high performance liquid chromatograph is 43 - 47 °C. Preferably, the column temperature of the chromatographic column 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. Preferably, the flow rate of the eluent in the high performance liquid chromatograph is 1.0 mL / min.

[0043] Preferably, the conditions of the evaporative light scattering detector in the high performance liquid chromatograph are as follows: nebulizer, drift tube temperature: 62 - 67 °C; nitrogen flow rate 1.4 - 1.8 L / min. Preferably, the conditions of the evaporative light scattering detector in the high performance liquid chromatograph are as follows: nebulizer, drift tube temperature: 65 °C; nitrogen flow rate 1.6 L / min. Description of the Drawings

[0044] Figure 1 It is the chromatogram of the eluent using different types of solid phase extraction columns in Example 1.

[0045] Figure 2 It is the chromatogram of the eluent using different types of sample loading solutions in Example 2.

[0046] Figure 3 It is the elution rate after using different types of eluents in Example 3.

[0047] Figures 4 - 6The reconstitution effects after using different types of reconstitution solutions in Example 4. Among them, Figure 4 is the reconstitution result diagram of 86% acetonitrile water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%, Figure 5 is the reconstitution result diagram of 86% acetone water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%, Figure 6 is the reconstitution result diagram 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 is the chromatogram after using different types of chromatographic columns in Example 5; Figure 8 is the chromatogram after using different types of mobile phases in Example 5.

[0049] Figure 9 is the chromatogram of isocratic elution with 80% acetonitrile water in Example 5; Figure 10 is the chromatogram of gradient elution in Example 5. Detailed implementation manners

[0050] To enable those skilled in the art to better understand the solution of 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 a part of the embodiments of the present invention, rather than all the embodiments.

[0051] The main instruments and devices used in the following examples are as follows: high-performance liquid chromatograph: Agilent 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 blowing concentrator: Biotage Turbo Vap LV. The main reagents and materials used are as follows: reference substances: L-fucitol reference standard (C6H14O5, CAS No.: 13074-06-1): purity ≥ 98%; trehalose reference standard (C12H22O11, CAS No.: 99-20-7): purity 99.8%; raffinose (C18H32O16, CAS No.: 512-69-6): purity 94.0%. Reagents: water (primary water), acetonitrile (chromatographic grade), methanol (chromatographic grade), acetone (chromatographic grade).

[0052] Example 1

[0053] Selection of solid phase extraction column:

[0054] In this example, HLB, C18, WAX, and NH were used respectively 2Detection was carried out using a [column name] / Si solid-phase extraction column, which was carried out respectively according to the following steps:

[0055] Step 1: In 80% acetonitrile-water (the volume ratio of acetonitrile to water is 80:20), add L-fucitol, trehalose, and raffinose standard products to obtain a mixed standard product solution;

[0056] Step 2: After activating the solid-phase extraction column (different solid-phase extraction columns are used according to the variables of this embodiment, namely HLB, C18, WAX, NH 2 / Si solid-phase extraction column), pass the mixed standard product solution in Step 1 through the activated solid-phase extraction column. Denote the solutions before and after entering the solid-phase extraction column as the sample loading solution and the eluate respectively. Nitrogen-blow the eluate to near dryness at 50 °C, re-dissolve it with 86% methanol aqueous solution, filter it through a 0.22 μm microporous filter membrane, and collect the filtrate in a sample injection vial for sample injection;

[0057] Step 3: Draw standard curves for L-fucitol, trehalose, and raffinose respectively; Drawing the standard curves for L-fucitol, trehalose, and raffinose can refer to Example 6 in the present invention (wherein, in the detection parameters of the high-performance liquid chromatograph, the elution program can refer to the gradient elution in Example 6 or the isocratic elution in this embodiment, and the other parameters are the same as those in this embodiment), but the specific standard curves may deviate specifically, and specific standard curves can be drawn according to the actual situation;

[0058] Step 4: Detect the eluate in Step 2 using a high-performance liquid chromatograph, substitute the obtained detection results into the standard curves, and obtain the contents of L-fucitol, trehalose, and raffinose in the eluate. And the chromatogram of the eluate is as Figure 1 shown.

[0059] It should be noted here that the HLB solid-phase extraction (60 mg / 3 mL) column was activated successively with 10 mL of methanol and 10 mL of water, the C18 solid-phase extraction column (500 mg / 3 mL) was activated successively with 10 mL of methanol and 10 mL of water, the WAX solid-phase extraction column (500 mg / 6 mL) was activated successively with 10 mL of methanol and 10 mL of water, and the NH 2 / Si solid-phase extraction column (1 g / 10 mL) was activated successively with 10 mL of methanol and 10 mL of water (the volume ratio of methanol to water is 1:1).

[0060] The detection conditions of the high-performance liquid chromatograph are as follows:

[0061] Chromatographic column: amino chromatographic column (4.6×250 mm, the particle size of the packing (filler) is 5 μm, and the diol-based bonded silica gel is used as the packing);

[0062] Sample volume: 20 μL;

[0063] Column temperature: 45 °C;

[0064] Flow rate: 1.0 mL / min;

[0065] Evaporative light scattering detector conditions: Nebulizer, drift tube temperature: 65 °C; Nitrogen flow rate 1.6 L / min;

[0066] Elution program: Mobile phase: Acetonitrile:Water (volume ratio) = 84:16 (During the pretreatment optimization process, standard substances were used, not involving sample matrix and without high separation requirements. Therefore, isocratic elution was used to enable rapid detection of the target compounds and save optimization time).

[0067] In addition, in the above step four, the contents of L-fucitol, trehalose, and raffinose in the effluent are shown in Table 1, and the recovery data calculated from the effluent are also shown in Table 1. Taking L-fucitol as an example, the recovery rate of L-fucitol = content of L-fucitol in the effluent / content of L-fucitol in the sample loading solution. The sample loading solution is the prepared mixed standard solution, and the calculation methods for the other two target compounds are the same; the higher the recovery rate, the worse the ability of the solid phase extraction column to retain the three target compounds.

[0068] Table 1 Contents and recovery data of L-fucitol, trehalose, and raffinose in the sample loading solution and effluent in step four of this example

[0069]

[0070] From Figure 1 and the results shown in Table 1, it can be seen that the recovery rates of the three target compounds (L-fucitol, trehalose, and raffinose) in the effluent from the HLB, C18, and WAX solid phase extraction columns are approximately 100%, indicating that all three target compounds flow out with the solvent, and the corresponding packing materials in the solid phase extraction column cannot achieve the purpose of retaining the target compounds, and the purification mechanism designed in the experiment cannot be realized. However, no target compound components were detected in the effluent from the NH 2 / 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 flow out with the solvent that may contain impurities such as fat-soluble vitamins, nucleotides, and water-soluble vitamins, and a better purification effect can be achieved. Therefore, the present invention selects the appropriate solid phase extraction column as the NH 2 / Si solid phase extraction column, that is, selecting NH 2 / Si SPE as the solid phase extraction column is more conducive to the separation of the three target compounds, and thus more conducive to the accuracy and sensitivity of the detection results.

[0071] Example 2

[0072] Selection of sample loading solution solvent:

[0073] NH 2 / Si solid-phase extraction column works on the principle that the amino functional group retains the target through hydrogen bonding and dipole-dipole interactions. Currently, the most commonly used solvents in the laboratory are mainly methanol, water, and acetonitrile. According to the analysis of the chemical properties of the solvents, it is known that methanol will have a hydrogen bonding interaction with the amino groups on the surface of the amino column, reducing the retention ability of the column for the target. Therefore, methanol is not suitable as the sample loading solution solvent. The hydrogen bonding interaction will be significantly weakened with the increase in the proportion of water, making it difficult to retain the target in the solid-phase extraction column. So, a high proportion of the aqueous phase is not suitable as the sample loading solution. Acetonitrile, due to its stable chemical structure, will not have a hydrogen bonding interaction with the amino groups, thus not affecting the retention of the target. Therefore, acetonitrile is suitable as the sample loading solution. However, since the target is easily soluble in water and insoluble in acetonitrile, if pure acetonitrile is selected as the sample loading solution solvent, the target cannot be dissolved in the solvent. Considering all these factors, in this experiment, acetonitrile-water is selected as the sample loading solution solvent. However, due to too high a proportion of the aqueous phase, it will weaken the retention ability of the NH 2 / Si solid-phase extraction column. If the proportion of the aqueous phase is too low, the target cannot be completely dissolved. Therefore, finding the appropriate acetonitrile-water ratio is crucial for the experiment.

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

[0075] Step 1, add L-fucitol, trehalose, and raffinose standard products into acetonitrile-water solutions with different volume ratios respectively to obtain mixed standard product solutions of different solvents. Here, according to the actual variables, the volume ratios of acetonitrile to water are 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 (the solid-phase extraction column uses NH 2 / Si solid-phase extraction column, and successively uses 10 mL of methanol and 10 mL of water (the volume ratio of methanol to water is 1:1) to activate the NH 2 / Si solid-phase extraction column), pass the mixed standard product solutions of different solvents in Step 1 through the activated solid-phase extraction column respectively, and record the solutions before and after entering the solid-phase extraction column as the sample loading solution and the effluent; blow the effluent to near dry at 50 °C with nitrogen, re-dissolve it with 86% methanol aqueous solution (the volume ratio of methanol to water is 86:14), filter it through a 0.22 μm microporous filter membrane, and collect the filtrate in a sample injection bottle for sample injection;

[0077] Step 3: Draw standard curves for L-fucitol, trehalose, and raffinose respectively. The drawing of the standard curves for L-fucitol, trehalose, and raffinose can refer to Example 6 in the present invention (wherein, in the detection parameters of the high performance liquid chromatograph, the elution program can refer to the gradient elution in Example 6 or the isocratic elution in Example 1, and the other parameters are the same as those in Example 1). However, the specific standard curves may deviate specifically, and specific standard curves can be drawn according to the actual situation;

[0078] Step 4: Detect the effluent in Step 2 using a high performance liquid chromatograph, and substitute the obtained detection results into the standard curve to obtain the contents of L-fucitol, trehalose, and raffinose in the effluent. The detection parameters of the high performance liquid chromatograph are the same as those in Example 1.

[0079] In the above steps, by detecting the content of the target substance in the effluent in Step 2, the content of the target substance retained in the NH 2 / Si solid phase extraction column can be calculated, and based on this, the retention ability of different sample loading solutions can be calculated. (The content of the target substance in the added standard - the content of the target substance in the effluent) / the content of the added standard * 100% = retention ability, and the content of the target substance in the added standard is the content of the target substance in the sample loading solution. When the retention ability reaches 100%, the target substance can be completely adsorbed on the solid phase extraction column to achieve the effect of separating the target substance from the impurities in the effluent.

[0080] After the detection through the above steps, the results are as Figure 2 shown, Figure 2 The results show that when 50% acetonitrile water (representing a volume ratio of acetonitrile to water of 50:50) is used as the solvent of the sample loading solution, the retention abilities of the three target substances are all below 40%. Continuing to increase the acetonitrile water ratio, the retention abilities of the three target substances are positively correlated with the increase of the acetonitrile water ratio. When 75% acetonitrile water is used as the solvent of the sample loading solution, the retention abilities of trehalose and raffinose reach the expected effects. However, at this ratio, the retention ability of L-fucitol is only 75%, which is not conducive to the simultaneous treatment of the three target substances. Continuing to increase the acetonitrile water ratio, the retention abilities of trehalose and raffinose do not decrease, and at the same time, the retention ability of L-fucitol can also reach the most ideal effect. In order to take into account the solubility of the target substances in the sample loading solution, 80% acetonitrile water is the best as the solvent of the sample loading solution.

[0081] Example 3

[0082] Selection of eluent:

[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, in this embodiment, ammoniated methanol, acetone, and methanol, which can react with the amino group in the solid-phase extraction column, are preferably used for the elution experiment. The specific steps are as follows:

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

[0085] Step 2: After activating the solid-phase extraction column (the solid-phase extraction column uses an NH 2 / Si solid-phase extraction column, and 10 mL of methanol and 10 mL of water (the volume ratio of methanol to water is 1:1) are used to activate the NH 2 / Si solid-phase extraction column in sequence), pass the mixed standard solution in Step 1 through the activated solid-phase extraction column. After the liquid in the column is completely drained, use the eluent for elution, collect all the eluents, blow to near dryness with nitrogen at 50 °C, redissolve with 86% methanol aqueous solution (the volume ratio of methanol to water is 86:14), filter through a 0.22 μm microporous filter membrane, and collect the filtrate in an injection vial for injection; according to the variables of this embodiment, different solvents (the same volume) are used as the eluent for the elution of the target substance during the elution process. Here, different solvents (different eluents) include ammoniated methanol, acetone, and methanol;

[0086] Step 3: Respectively plot the standard curves for L-fucitol, trehalose, and raffinose; Refer to Example 6 in the present invention for plotting the standard curves for L-fucitol, trehalose, and raffinose (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 the same as those in Example 1), but the specific standard curves may deviate specifically, and specific standard curves can be plotted according to the actual situation;

[0087] Step 4: Detect the filtrate in Step 2 using a high-performance liquid chromatograph, and substitute the obtained detection results into the standard curve to obtain the contents of L-fucitol, trehalose, and raffinose in the eluent respectively. The detection parameters of the high-performance liquid chromatograph are the same as those in Example 1.

[0088] In the above steps, by detecting the content of the target substance in the eluent, the content of the target substance retained in the eluent can be calculated, and based on this, the elution ability of different eluents can be calculated. (The content of the target substance in the eluent) / the content of the added standard * 100% = elution ability (elution rate), and the content of the added standard is the content of the standard in Step 1. When the elution ability is close to 100%, the target substance can almost all flow out of the solid-phase extraction column to meet the detection requirements.

[0089] After the above steps of detection and related calculations, the results are as Figure 3 shown Figure 3 The results show that ammoniated methanol can elute 54.6% of L-fucitol and 79.4% of raffinose, while the elution rate of trehalose is only 12.2%; acetone can elute 99.8% of L-fucitol and 69.9% of trehalose, while the elution rate of raffinose is basically 0; methanol can elute 98.9% of L-fucitol, 99.6% of trehalose and 99.4% of raffinose. Therefore, methanol has the highest elution rate for all three target substances, that is, it shows excellent and balanced elution effects on the three target substances.

[0090] Example 4

[0091] Selection of reconstitution solution:

[0092] In this example, different types of complex solutions are used as variables to explore the reconstitution effects of different types of complex solutions. The specific steps are as follows:

[0093] Take 4 g of sample with a sugar content of 0.5% (mass fraction) (the sugar content is 4 g * 0.5% = 0.02 g). After diluting the 4 g sample to 10 ml with water, take 1.5 ml and pass it through a solid-phase extraction column (NH 2 / Si solid-phase extraction column, the NH 2 / Si solid-phase extraction column is activated with 10 mL of methanol and 10 mL of water in sequence). After all the liquid in the column is drained, elute with methanol, collect all the eluates, blow to near dry at 50 °C with nitrogen, reconstitute with a complex solution, make the volume up to 1 mL, filter through a 0.22 μm microporous membrane, and collect the filtrate; the sugar content in the solid-phase extraction column should be 0.02 g * 1.5 ml / 10 ml = 0.003 g. When reconstituting a 0.5% sugar content sample, 1 ml of the complex solution needs to reconstitute 0.003 g of sugar.

[0094] To more conveniently and intuitively observe the scene, this experiment uses 10 ml of the complex solution to reconstitute 0.03 g of sugar to simulate the pre-treatment process of 1 ml of the complex solution reconstituting a 0.5% sugar content sample. Weigh 0.25 g of sucrose, dilute it to 10 ml with water, mix well and transfer 1.2 ml to make the sugar content in the aqueous phase 0.03 g, and add 8.8 mL of acetonitrile, acetone, and methanol respectively to simulate the reconstitution phenomenon of a 5% sugar content sample reconstituted with a 14% aqueous phase and 86% organic phase. If stratification or precipitation is observed, it proves that the reconstitution effect cannot meet the experimental requirements and subsequent detections cannot be carried out. By analogy, observe the reconstitution scene with sugar contents of 1.0%, 1.5%, 2.0%, 5.0%, 10.0%, and 15.0%.

[0095] The reconstitution results are asFigure 4 , 5 As shown in Figure 6, Figure 4 is the reconstitution result diagram of 86% acetonitrile-water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%; Figure 5 is the reconstitution result diagram of 86% acetone-water with sugar contents of 0.5%, 1.0%, 1.5%, and 2.0%; Figure 6 is the reconstitution result diagram of 86% acetonitrile-water with sugar contents of 0.5%, 1.0%, 1.5%, 2.0%, 5.0%, 10.0%, and 15.0%. It can be seen from Figure 4 that when reconstituting the sample with a sugar content of 0.5% using acetonitrile-water, obvious stratification occurred, and as the sugar content increased, the amount of the acetonitrile-poor phase in the lower layer also increased. Acetonitrile-water is not suitable as a reconstitution solution. Subsequently, the laboratory tried to use acetone-water for reconstitution, Figure 5 and the results showed that the sample with a sugar content of 0.5% had a good reconstitution effect under the reconstitution of acetone-water, but obvious solids precipitated when the sugar content of the sample was 1.0%. The reconstitution ability was negatively correlated with the increase in sugar content. Obviously, acetone-water is only suitable as a reconstitution solution for samples with low sugar contents. To find a reconstitution solution with better reconstitution ability, the laboratory then selected methanol-water for testing (isopropanol and ethanol in alcohol compounds have relatively high viscosities, which will cause a significant increase in column pressure and are not suitable as alternative reconstitution solutions). By observing Figure 6 the phenomenon, it was found that even when reconstituting the sample with a sugar content as high as 15% using methanol-water, no sugar precipitation and stratification occurred, and the reconstitution ability of methanol-water fully met the detection requirements of the experiment. Thus, it can be seen that methanol-water is an excellent choice for the reconstitution solution in this experiment.

[0096] Example 5

[0097] (1) Optimization of instrument conditions:

[0098] First, in this example, different types of chromatographic columns were used 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 (the volume ratio of methanol to water is 86:14) as the solvent, prepare a standard mixed injection solution of L-fucitol standard, trehalose standard, and raffinose standard (containing L-fucitol 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 sample solution into the high-performance liquid chromatograph for detection; in this step, according to the variables of the first aspect of this embodiment, adjust different types of chromatographic columns in the high-performance liquid chromatograph (including C18 chromatographic column, diol-based chromatographic column, and amino chromatographic column. The three chromatographic columns are all with a diameter of (25 cm), an inner diameter of (4.6 mm), and a filler particle size of (5 μm) for detection. The detection conditions of the remaining high-performance liquid chromatograph are the same as those in Example 1).

[0101] The test results are as Figure 7 shown. The target substance in the C18 chromatographic column has been completely detected at 2.5 min, and this chromatographic column has no retention and aggregation effect on the target substance. Both the diol-based chromatographic column and the amino chromatographic column have good separation and retention effects on the 3 target substances and can meet the detection requirements. By carefully comparing the chromatograms of the two, it can be found that there is no obvious difference in the peak area of L-fucitol whether using the amino chromatographic column or the diol-based chromatographic column, but the peak area of trehalose and raffinose detected using the diol-based chromatographic column is only about one-third of the peak area detected using the amino chromatographic column. The higher the detection peak area of the same concentration compound, the higher the sensitivity of the instrument, which can reduce the detection limit, improve the accuracy of the analysis result, and increase the reliability and repeatability of the data. By comparison, the amino chromatographic column is the better choice in this experiment. Therefore, choosing the amino chromatographic column as the chromatographic column has better advantages and is more conducive to improving the overall accuracy and sensitivity of the detection results.

[0102] (2) Selection of mobile phase:

[0103] In the second aspect, this embodiment takes different mobile phases as variables to explore the detection effects of different mobile phases. The specific steps are as follows:

[0104] Step 1: Using methanol-water (the volume ratio of methanol to water is 86:14) as the solvent, prepare a standard mixed sample solution (containing L-fucitol 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) from the L-fucitol standard, trehalose standard, and raffinose standard.

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

[0106] The test results are as Figure 8As shown, using methanol-water as the mobile phase cannot effectively separate the three target substances, while using acetonitrile-water as the mobile phase can effectively separate the three target substances. This is because methanol forms hydrogen bonds with the amino functional groups of the amino chromatographic column, reducing the retention ability of the chromatographic column for the target substances, resulting in the detection of the target substances within the first 3 minutes and failing to achieve the effect of effectively separating the target substances. Due to its chemical molecular structure, acetonitrile is not likely to react with the packing material inside the amino chromatographic column, and its characteristics of low viscosity and low surface tension help improve the resolution and sensitivity of liquid chromatography, performing well during the detection process, which can further promote the effective separation of the three target substances, and thus is more conducive to improving the overall accuracy and sensitivity of the detection results.

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

[0108] Thirdly, in this embodiment, the mobile phase elution program is also optimized. Since the target substances are similar in properties to the common fructose, glucose, sucrose, maltose, and lactose in beverages, the laboratory optimized the mobile phase elution program with a key consideration of whether the target substances and fructose, glucose, sucrose, maltose, and lactose can be effectively analyzed. First, the separation ability of isocratic elution with 80% acetonitrile-water was explored, and the results are as Figure 9 shown, Figure 9 It was found that the retention times of the characteristic peaks of trehalose overlap with those of lactose and maltose (lactose and maltose overlap), and the separation effect fails to reach the expected effect.

[0109] Furthermore, the laboratory then explored the use of gradient elution mode. During the verification process, it was found that the amino chromatographic column is sensitive to changes in the mobile phase. If the change speed of the mobile phase is too fast, it will cause too large baseline fluctuations, and the change of the mobile phase is not suitable to be too frequent and intense. After repeated verification, it was found that 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), 39.00 - 40.00 min (14% B) as the elution program has a good separation effect, and all three target substances can be effectively separated from the five common sugars in the experiment and the beverage. The separation effect is as Figure 10 shown, and the elution program meets the detection requirements. Therefore, using the above elution program is more conducive to improving the overall accuracy and sensitivity of the detection results.

[0110] Example 6

[0111] Quantitation limit, standard curve, accuracy and precision:

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

[0113] S1. Extraction: First, dissolve the sample with water (weigh 4 g of the sample into a 10 mL volumetric flask and make up to the mark with water). After centrifugation at a speed of 4000 r / min for 3 min, take 1.5 mL of the first supernatant into a 15 mL centrifuge tube, then add 6 mL of acetonitrile, vortex to mix evenly, and ultrasonicate for 10 min, and collect the resulting mixed solution. In this example, the volume ratio of the first supernatant to acetonitrile is recorded as 20:80 (since the sugar-containing sample is mainly water and the sugar content is very low, the first supernatant can be approximately regarded as all water).

[0114] S2. Purification: Transfer the mixed solution obtained in S1 into an activated NH 2 / Si solid-phase extraction column (activate the NH 2 / Si solid-phase extraction column successively with 10 mL of methanol + 10 mL of water (the volume ratio of methanol to water is 1:1). The specifications of the NH 2 / Si solid-phase extraction columns are all 1 g / 10 mL). Then, add 2 mL of 80% acetonitrile-water (the volume ratio of acetonitrile to water is 80:20) at a liquid level of 2.0 mm. After the liquid in the column is completely drained, elute with 25 mL of methanol, collect all the eluates, blow to near dry under nitrogen at 50 °C, re-dissolve with 86% methanol aqueous solution (the volume ratio of methanol to water is 86:14), make up to 1 mL, filter through a 0.22 μm microporous filter membrane, and collect the filtrate for injection.

[0115] S3. Prepare a standard curve for L-fucitol, trehalose, and raffinose;

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

[0117] Table 2 Specific program of gradient elution

[0118] Time / min A / ％ B / ％ 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] Among them, the preparation steps of the standard curve for L-fucitol, trehalose, and raffinose in S3 are as follows:

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

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

[0122] Standard mixed working solution: Accurately pipette 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, and 400 μL respectively, and make up to 1 mL with a methanol-aqueous solution (volume ratio 86:14). Prepare a standard mixed working solution (containing L-fucitol concentrations of 0.04 mg / mL, 0.08 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, trehalose concentrations of 0.025 mg / mL, 0.05 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and raffinose concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL)

[0123] Plot the standard curve: Inject the above L-fucitol, trehalose, and raffinose solutions with different concentrations into a high-performance liquid chromatograph for testing respectively (the test conditions of the high-performance liquid chromatograph can be the same as those in Example 1; or the isocratic elution in Example 1 can be changed to the gradient elution in this example, and the other detection conditions are the same as those in Example 1). Plot the standard curve with the peak area of the target substance as the ordinate and the concentration of the corresponding standard mixed working solution as the abscissa. The relevant information of the obtained standard curve is shown in Table 3:

[0124] Table 3 Relevant information of the standard curve

[0125]

[0126] After drawing the relevant standard curves in S3, perform the sample detection steps in S4. Conduct a spiked recovery experiment at three levels (quantitation limit, twice the quantitation limit, and ten times the quantitation limit) with six replicates for negative samples (samples without the target substance to be detected, also known as blank samples) to verify the accuracy and precision. The experimental results show that the recovery rate of L-fucitol is 95.63 - 102.50%, and the RSD value is 1.19 - 2.45%; the recovery rate of trehalose is 92.00 - 102.00%, and the RSD value is 2.27 - 2.56%; the recovery rate of raffinose is 96.00 - 103.10%, and the RSD value is 2.17 - 2.90%. The accuracy and precision of the method meet the requirements of the GB / T 27404-2008 standard. The specific values are shown in Table 4. The above results indicate that the detection method provided by the present invention has high accuracy and precision. Among them, taking the quantitation limit of the spiked level in Table 4 as 0.08 g / kg as an example, 0.08 g / kg represents the spiked concentration, that is, the detection state when simulating 0.08 g of the target substance in 1 kg of the sample.

[0127] Table 4 Verification Results of Accuracy and Precision

[0128]

[0129] Example 7

[0130] Re - validation of different samples:

[0131] To verify the universality of the detection method provided by the present invention for different beverages, several beverages that are prone to adding sweeteners on the market (carbonated beverages, vitamin functional beverages, plant protein beverages, and tea beverages) were selected to conduct spiked recovery experiments at the quantitation limit level, and the accuracy parameters were re-verified. The specific test steps refer to the method of Example 6. The final verification results are shown in Table 5. The results show that the spiked recovery rates of several matrices meet the requirements of the GB / T 27404-2008 standard, indicating that the detection method provided by the present invention is suitable for detecting different types of beverages and has a wide range of applications.

[0132] Table 5 Spiked Recovery Results of Different Samples

[0133]

[0134]

[0135] In summary, the present invention uses solid-phase extraction for pretreatment and determines the contents of L-fucitol, trehalose, and raffinose in beverages by high-performance liquid chromatography. Moreover, the established detection method is applied to different types of beverages, and the detection effect is good. This method has high precision, good accuracy, short time consumption, and easy operation, and can detect 3 kinds of sweeteners at one time, providing a new detection technology and basis for the simultaneous detection of multiple target substances of sugars and sugar alcohols with different chemical structures.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A method for detecting the content of L-fucose, trehalose and raffinose in a beverage, characterized in that: The steps include: S1. Extraction, first dissolving the sample in water, centrifuging, taking a first supernatant, then adding a first organic solvent to the first supernatant and mixing them 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, transferring the mixed solution into an activated amino solid phase extraction column, and after the liquid in the column is completely discharged, eluting with a second organic solvent, collecting all the eluate, removing the solvent in the eluate, re-dissolving, filtering, and collecting the filtrate; the second organic solvent includes methanol; S3. Prepare standard curves for L-fucose, trehalose, and raffinose; S4. injecting the filtrate into a high performance liquid chromatograph for detection, and substituting the detection result into the standard curve to determine the content of the L-fucose, the trehalose, and the raffinose in the sample.

2. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage as claimed in claim 1, characterized in that: In the above S1, during the centrifugal treatment, the centrifugal speed is 3500-4500 r / min, and the centrifugal time is 2-5 min.

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

4. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage as claimed in claim 1, characterized in that: In S2, after the mixed solution is transferred into the activated amino solid phase extraction column, a mixed solvent is added at a liquid level of 1.5 to 2.5 mm above the column. The mixed solvent includes water and acetonitrile, and the volume ratio of water to acetonitrile is 10 to 30:70 to 90.

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

6. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage as claimed in claim 1, characterized in that: In S2, during the re-dissolution process, the re-dissolution solution used includes methanol and water; and the volume ratio of methanol to water is 80-92:8-20.

7. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage as claimed in claim 1, characterized in that: In S3, the R of the linear regression equation for the standard curves of L-fucose, trehalose, and raffinose is 2 The values ​​are not less than 0.

99.

8. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage according to claim 7, characterized in that: The specific operation of preparing the standard curve of the L-fucose, the trehalose, and the raffinose in S3 is as follows: Step 1, preparing different standard mixed working solutions of the L-fucitol, the trehalose, and the raffinose, wherein the concentration points of the L-fucitol include 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 the trehalose include 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 the raffinose include 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL; Step 2: draw the standard curve with the measured target peak area as the ordinate and the corresponding standard mixed working solution concentration as the abscissa.

9. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage according to claim 1, characterized in that: In said S4, the chromatographic column of the high performance liquid chromatograph comprises an amino chromatographic column.

10. The method for detecting the content of L-fucitol, trehalose and raffinose in a beverage according to claim 1, characterized in that: In S4, the elution process of the high performance liquid chromatograph is gradient elution, and the eluent in the elution process includes mobile phase A and mobile phase B; the mobile phase A is acetonitrile, and the mobile phase B is water, and the specific elution procedure is as follows:

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