Method for detecting nine glycoside sweeteners in surimi product by isotope internal standard method

Abstract

The invention discloses a method for detecting nine glycoside sweeteners in a surimi product through an isotope internal standard method, belongs to the technical field of compound detection, and particularly relates to a method for detecting nine glycoside sweeteners in a surimi product. The method comprises the following steps: extracting and purifying a supernatant with a purifying agent, finally passing through a membrane to obtain a surimi treatment solution, and detecting the content of the glycoside sweeteners in the surimi treatment solution by adopting UPLC-MS / MS. According to the method, isotope labeled [U-13C]-stevioside is taken as an internal standard, a series of mixed standard solutions of nine glycoside sweeteners with a series of concentrations are prepared, a standard curve is drawn, and the content of the glycoside sweeteners in the surimi treatment solution is determined. The method can be used for calculating the content of the glycoside sweetening agent in the surimi product. The method disclosed by the invention has a good detection effect on nine glycoside sweeteners in the surimi product.

Description

Technical Field

[0001] This invention belongs to the field of compound detection technology, specifically relating to a method for detecting nine glycoside sweeteners in surimi products using the isotope internal standard method. Background Technology

[0002] Glycoside sweeteners are used in aquatic products to provide sweetness while reducing the product's calorie content. As alternative sugars or low-calorie sweeteners, they satisfy consumers' sweetness needs while helping them control calorie intake. To increase economic benefits, some seafood vendors add multiple sweeteners to improve taste; however, the amount added directly affects consumers' health, and the resulting safety issues are becoming increasingly prominent. Detecting and monitoring sweeteners in aquatic products has become a crucial task for safety monitoring departments. Therefore, developing a rapid, accurate, and sensitive method for detecting sweeteners in aquatic products is essential to ensuring their quality and safety.

[0003] Currently, common methods for detecting sweeteners in aquatic products include high-performance liquid chromatography-ultraviolet (HPLC-UV) detection, capillary gas chromatography, ultraviolet spectrophotometry, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Among these, capillary gas chromatography and ultraviolet spectrophotometry are generally used to determine single or two sweeteners, and their detection sensitivity needs improvement. HPLC-UV detection, due to its inherent limitations, suffers from low sensitivity, long detection time, poor resolution, and susceptibility to interference from impurities, leading to false positives. This not only increases the workload of laboratory operators but also makes it difficult to meet the needs and development trends of simultaneous detection of multiple components.

[0004] Compared with the methods mentioned above, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) offers advantages such as strong separation capability, reliable qualitative analysis results, low detection limit, fast analysis time, and high degree of automation. HPLC-MS / MS leverages the complementary strengths of chromatography and mass spectrometry, combining the high separation capability of chromatography for complex samples with the high selectivity, high sensitivity, and ability to provide relative molecular mass and structural information of mass spectrometry. In recent years, there have been some reports on using this method to detect sweeteners in various foods. However, these reports use only one detection parameter and are mostly aimed at alcoholic beverages, dairy products, and condiments. There are no reports on the simultaneous detection of nine sweeteners in aquatic products, namely rubusoside, steviolbioside, dulcoside A, rebaudioside B, stevia, rebaudioside F, rebaudioside C, rebaudioside A, and rebaudioside D. Summary of the Invention

[0005] The purpose of this invention is to provide an isotope internal standard method for detecting nine glycoside sweeteners in surimi products, which has a low detection limit, high recovery rate, and small relative standard deviation.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for detecting nine glycoside sweeteners in surimi products includes: pretreating the surimi products to obtain a surimi treatment solution, and using UPLC-MS / MS to detect the nine glycoside sweeteners in the surimi treatment solution; the pretreatment uses a purifying agent, including MgSO4, with the amount of MgSO4 used being 2-10 wt% of the surimi products. This invention utilizes ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to simultaneously detect nine glycoside sweeteners in surimi products. It offers high sensitivity, good stability, fast analysis time, and a high degree of automation, providing technical support for ensuring the safety and quality of surimi products.

[0007] Preferably, the method further includes preparing a mixed standard solution using the isotope internal standard method to construct a standard working curve, wherein the isotope internal standard reagent is [U-13C]-stevioside.

[0008] Preferably, the purifying agent also includes PSA, and the amount of PSA used is 0.3-2 wt% of the surimi product.

[0009] Preferably, the purifying agent also includes C18, and the amount of C18 used is 0.6-4 wt% of the surimi product.

[0010] Preferably, the purifying agent also includes porous biochar, and the amount of porous biochar used is 0.1-1 wt% of the surimi product.

[0011] More preferably, the porous biochar is prepared from cyclodextrin derivatives, chitosan, and citric acid. The cyclodextrin derivatives are formed by reacting p-toluenesulfonyl cyclodextrin with an amino compound, including 2-aminopyrrole. The porous biochar contains a carbonized structure resulting from the calcination of the cyclodextrin derivatives, chitosan, and citric acid. The cyclodextrin derivatives possess a 2-aminopyrrole structure. Therefore, the porous biochar contains the structures of cyclodextrin derivatives, chitosan, citric acid, and 2-aminopyrrole. This invention uses a mixture of porous biochar, magnesium sulfate, and PSA as a purifying agent, which can reduce interfering substances in the supernatant of surimi products, improve the detection effect of nine glycoside sweeteners in surimi products, achieve high recovery rates for glycoside sweeteners in surimi products, and exhibit a small relative standard deviation for the detection of glycoside sweeteners in surimi products.

[0012] Preferably, the pretreatment process first involves extraction with an aqueous methanol solution, followed by treatment with a purification agent, and finally membrane filtration.

[0013] Preferably, the amount of surimi product used is 10-30 wt% of the methanol aqueous solution.

[0014] Preferably, the methanol content in the methanol-water solution is 10-60 vol.

[0015] Preferably, the nine glycoside sweeteners are steviol glycoside, ribobadiol A, ribobadiol B, ribobadiol C, ribobadiol D, ribobadiol F, durqueside A, steviol disaccharide, and stevia glycoside.

[0016] Preferably, in the preparation of the surimi treatment solution, the surimi product is mixed with a methanol aqueous solution, shaken at 20-40℃ for 5-30 min, sonicated for 5-30 min, and then centrifuged at 3000-9000 r / min for 3-10 min. The supernatant is taken, a purifying agent is added for extraction and purification, and after shaking and centrifugation, it is filtered through a membrane to obtain the surimi treatment solution.

[0017] More preferably, in the preparation of the surimi treatment solution, the methanol content in the methanol aqueous solution is 10-60 vol.

[0018] More preferably, in the preparation of the surimi treatment solution, the amount of surimi product used is 10-30 wt% of the methanol aqueous solution.

[0019] More preferably, in the preparation of the surimi treatment liquid, the purifying agent includes MgSO4, PSA and C18, wherein the amount of MgSO4 used is 2-10 wt% of the surimi product, the amount of PSA used is 0.3-2 wt% of the surimi product, and the amount of C18 used is 0.6-4 wt% of the surimi product.

[0020] More preferably, in the preparation of the surimi treatment liquid, the purifying agent includes MgSO4, PSA and porous biochar, and the amount of porous biochar used is 0.1-1 wt% of the surimi product.

[0021] Preferably, the preparation of porous biochar includes the preparation of p-toluenesulfonyl cyclodextrin and the preparation of cyclodextrin derivatives.

[0022] Preferably, in the preparation of p-toluenesulfonyl cyclodextrin, β-cyclodextrin is added to deionized water and mixed, then p-toluenesulfonyl chloride is added, and the mixture is stirred at 20-40℃ for 4-16 h. Then, an alkaline solution is added to obtain a suspension, which is stirred for 10-60 min. The suspension is filtered, the pH is adjusted to 8-9, and then the mixture is allowed to stand at 0-10℃ for 5-20 h to precipitate. The precipitate is filtered, washed, and freeze-dried to obtain p-toluenesulfonyl cyclodextrin.

[0023] More preferably, in the preparation of p-toluenesulfonyl cyclodextrin, the amount of β-cyclodextrin used is 2-10 wt% of deionized water.

[0024] More preferably, in the preparation of p-toluenesulfonyl cyclodextrin, the amount of p-toluenesulfonyl chloride used is 20-40 wt% of β-cyclodextrin.

[0025] More preferably, in the preparation of p-toluenesulfonyl cyclodextrin, the alkaline solution is a sodium hydroxide solution, the sodium hydroxide content in the sodium hydroxide solution is 1-5 mmol / L, and the amount of sodium hydroxide solution used is 10-30 wt% of deionized water.

[0026] Preferably, in the preparation of cyclodextrin derivatives, p-toluenesulfonyl cyclodextrin and an amino compound are added to a mixture of triethanolamine and deionized water, and the mixture is heated under reflux for 12-48 hours in an inert gas atmosphere. After the reaction is completed, the solvent is removed by vacuum distillation, and then anhydrous ethanol is added. The mixture is then refrigerated at 0-10°C for 6-24 hours to precipitate the precipitate. After filtration and drying, the cyclodextrin derivative is obtained.

[0027] More preferably, in the preparation of the cyclodextrin derivative, triethanolamine and deionized water are mixed in a volume ratio of 1:1-2.

[0028] More preferably, in the preparation of cyclodextrin derivatives, the amount of p-toluenesulfonylcyclodextrin used is 5-15 wt% of the mixture.

[0029] More preferably, in the preparation of the cyclodextrin derivative, the amino compound is 2-aminopyrrole, and the amount of 2-aminopyrrole used is 20-40 wt% of p-toluenesulfonyl cyclodextrin.

[0030] More preferably, in the preparation of the cyclodextrin derivative, nitrogen is used as the inert gas, and the reflux temperature is controlled at 100°C. The amount of anhydrous ethanol used is the same as the amount of the mixture.

[0031] Preferably, in the preparation of porous biochar, cyclodextrin derivatives and chitosan are added to citric acid solution and mixed evenly, the water is evaporated, and calcined at 700-900℃ for 1-5 hours in an inert gas atmosphere, and then cooled to obtain porous biochar.

[0032] More preferably, in the preparation of porous biochar, the citric acid content in the citric acid solution is 0.5-2 wt%.

[0033] More preferably, in the preparation of porous biochar, the amount of cyclodextrin derivative used is 10-30 wt% of citric acid solution.

[0034] More preferably, in the preparation of porous biochar, the amount of chitosan used is 20-40 wt% of the cyclodextrin derivative, and the inert gas is nitrogen.

[0035] More preferably, pentaerythritol tetracitrate can be added during the preparation of porous biochar, with the amount of pentaerythritol tetracitrate being 1-10 wt% of the cyclodextrin derivative. After further adding pentaerythritol tetracitrate in this invention, the porous biochar possesses the structure of cyclodextrin derivative, chitosan, citric acid, 2-aminopyrrole, and pentaerythritol tetracitrate. Under the co-extraction and purification of porous biochar with magnesium sulfate and PSA, the detection effect of nine glycoside sweeteners in surimi products can be improved, resulting in a high recovery rate and a small relative standard deviation for the detection of glycoside sweeteners in surimi products.

[0036] Preferably, the glycoside sweeteners in the surimi processing solution are detected by UPLC-MS / MS.

[0037] This invention involves mixing surimi products with a methanol-water solution, centrifuging to remove the supernatant, purifying the supernatant with a purifying agent, and finally passing it through a membrane to obtain a surimi-treated solution. The surimi-treated solution is then analyzed using UPLC-MS / MS to detect the content of glycoside sweeteners. This invention uses isotopically labeled [U-13C]-stevioside as an internal standard to prepare a series of mixed standard solutions of nine glycoside sweeteners at various concentrations, and plots a standard curve to calculate the glycoside sweetener content in the surimi products. This invention uses porous biochar as a purifying agent, prepared from cyclodextrin derivatives, which are formed by reacting p-toluenesulfonylcyclodextrin with amino compounds, including 2-aminopyrrole. Therefore, this method offers the following advantages: high recovery rate and small relative standard deviation for glycoside sweeteners. Thus, this invention provides a method for detecting nine glycoside sweeteners in surimi products using an isotopic internal standard method, characterized by low detection limits, high recovery rates, and small relative standard deviations. Attached Figure Description

[0038] Figure 1 This is a SEM image of porous biochar. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0041] Example 1: A method for detecting nine types of glycoside sweeteners in surimi products Preparation of surimi treatment solution: Surimi products were mixed with methanol-water solution, shaken at 30℃ for 10 min, sonicated for 10 min, and then centrifuged at 6000 r / min for 5 min. The supernatant was collected, and a purifying agent was added for extraction and purification. After shaking and centrifugation, the mixture was filtered through a membrane to obtain the surimi treatment solution. The methanol content in the methanol-water solution was 50 vol%, and the amount of surimi products used was 20 wt% of the methanol-water solution. The purifying agents included MgSO4, PSA, and C18. The amount of MgSO4 used was 4 wt% of the surimi products, the amount of PSA used was 0.6 wt% of the surimi products, and the amount of C18 used was 1.2 wt% of the surimi products.

[0042] Detection of glycoside sweeteners: UPLC-MS / MS was used to detect glycoside sweeteners in the surimi processing solution.

[0043] Example 2: A method for detecting nine types of glycoside sweeteners in surimi products Preparation of p-toluenesulfonyl cyclodextrin: β-cyclodextrin was added to deionized water and mixed, then p-toluenesulfonyl chloride was added. The mixture was stirred at 30°C for 8 hours. An alkaline solution was then added to obtain a suspension, which was stirred for 30 minutes. The suspension was filtered, and the pH was adjusted to 8.5. The suspension was then allowed to stand at 5°C for 10 hours to precipitate. The precipitate was filtered, washed, and freeze-dried to obtain p-toluenesulfonyl cyclodextrin. The amount of β-cyclodextrin used was 5 wt% of deionized water, the amount of p-toluenesulfonyl chloride used was 30 wt% of β-cyclodextrin, and the alkaline solution was sodium hydroxide solution with a sodium hydroxide concentration of 3 mmol / L, and the amount of sodium hydroxide solution used was 20 wt% of deionized water.

[0044] Preparation of cyclodextrin derivatives: p-Toluenesulfonyl cyclodextrin and an amino compound were added to a mixture of triethanolamine and deionized water. The mixture was heated under reflux for 24 hours under an inert gas atmosphere. After the reaction was complete, the solvent was removed by vacuum distillation. Then, anhydrous ethanol was added, and the mixture was refrigerated at 5°C for 12 hours to precipitate the precipitate. The precipitate was filtered and dried to obtain the cyclodextrin derivatives. The triethanolamine and deionized water in the mixture were mixed in a 1:1 volume ratio. The amount of p-toluenesulfonyl cyclodextrin used was 10 wt% of the mixture. The amino compound was 2-aminopyrrole, and the amount of 2-aminopyrrole used was 30 wt% of p-toluenesulfonyl cyclodextrin. Nitrogen was used as the inert gas, and the reflux temperature was controlled at 100°C. The amount of anhydrous ethanol used was the same as the amount of the mixture.

[0045] Preparation of porous biochar: Cyclodextrin derivatives and chitosan were added to a citric acid solution and mixed evenly. The water content was evaporated, and the mixture was calcined at 800℃ for 3 hours in an inert gas atmosphere. After cooling, porous biochar was obtained. The citric acid content in the citric acid solution was 1 wt%, the amount of cyclodextrin derivative used was 20 wt% of the citric acid solution, the amount of chitosan used was 30 wt% of the cyclodextrin derivative, and the inert gas was nitrogen.

[0046] Preparation of surimi treatment solution: Surimi products were mixed with methanol-water solution, shaken at 30℃ for 10 min, sonicated for 10 min, and then centrifuged at 6000 r / min for 5 min. The supernatant was collected, and a purifying agent was added for extraction and purification. After shaking and centrifugation, the mixture was filtered through a membrane to obtain the surimi treatment solution. The methanol content in the methanol-water solution was 50 vol%, and the amount of surimi products used was 20 wt% of the methanol-water solution. The purifying agents included MgSO4, PSA, and porous biochar. The amount of MgSO4 used was 4 wt% of the surimi products, the amount of PSA used was 0.6 wt% of the surimi products, and the amount of porous biochar used was 0.8 wt% of the surimi products.

[0047] Detection of glycoside sweeteners: UPLC-MS / MS was used to detect glycoside sweeteners in the surimi processing solution.

[0048] Example 3: A method for detecting nine types of glycoside sweeteners in surimi products The difference between this embodiment and Example 2 lies in the preparation of the surimi treatment solution.

[0049] Preparation of surimi treatment solution: Surimi products were mixed with methanol-water solution, shaken at 30℃ for 10 min, sonicated for 10 min, and then centrifuged at 6000 r / min for 5 min. The supernatant was collected, and a purifying agent was added for extraction and purification. After shaking and centrifugation, the mixture was filtered through a membrane to obtain the surimi treatment solution. The methanol content in the methanol-water solution was 50 vol%, and the amount of surimi products used was 20 wt% of the methanol-water solution. The purifying agents included MgSO4, PSA, and porous biochar. The amount of MgSO4 used was 4 wt% of the surimi products, the amount of PSA used was 0.6 wt% of the surimi products, and the amount of porous biochar used was 0.2 wt% of the surimi products.

[0050] Example 4: A method for detecting nine types of glycoside sweeteners in surimi products The difference between this embodiment and Example 2 lies in the preparation of porous biochar.

[0051] Preparation of porous biochar: Cyclodextrin derivatives and chitosan were added to a citric acid solution and mixed evenly. The water content was evaporated, and the mixture was calcined at 800℃ for 3 hours in an inert gas atmosphere. After cooling, porous biochar was obtained. The citric acid content in the citric acid solution was 1 wt%, the amount of cyclodextrin derivative used was 20 wt% of the citric acid solution, the amount of chitosan used was 30 wt% of the cyclodextrin derivative, the amount of pentaerythritol tetracitrate used was 6 wt% of the cyclodextrin derivative, and nitrogen was used as the inert gas.

[0052] Example 5: A method for detecting nine types of glycoside sweeteners in surimi products The difference between this embodiment and Example 4 lies in the preparation of the surimi treatment solution.

[0053] Preparation of surimi treatment solution: Surimi products were mixed with methanol-water solution, shaken at 30℃ for 10 min, sonicated for 10 min, and then centrifuged at 6000 r / min for 5 min. The supernatant was collected, and a purifying agent was added for extraction and purification. After shaking and centrifugation, the mixture was filtered through a membrane to obtain the surimi treatment solution. The methanol content in the methanol-water solution was 50 vol%, and the amount of surimi products used was 20 wt% of the methanol-water solution. The purifying agents included MgSO4, PSA, and porous biochar. The amount of MgSO4 used was 4 wt% of the surimi products, the amount of PSA used was 0.6 wt% of the surimi products, and the amount of porous biochar used was 0.2 wt% of the surimi products.

[0054] Comparative Example 1: A method for detecting nine glycoside sweeteners in surimi products The difference between this comparative example and Example 2 lies in the preparation of the surimi treatment solution.

[0055] Preparation of surimi treatment solution: Surimi products were mixed with methanol-water solution, shaken at 30℃ for 10 min, sonicated for 10 min, and then centrifuged at 6000 r / min for 5 min. The supernatant was collected, and a purifying agent was added for extraction and purification. After shaking and centrifugation, the mixture was filtered through a membrane to obtain the surimi treatment solution. The methanol content in the methanol-water solution was 50 vol%, and the amount of surimi products used was 20 wt% of the methanol-water solution. The purifying agents included MgSO4, PSA, and porous biochar. The amount of MgSO4 used was 4 wt% of the surimi products, the amount of PSA used was 0.6 wt% of the surimi products, and the amount of porous biochar used was 0.08 wt% of the surimi products.

[0056] Comparative Example 2: A method for detecting nine glycoside sweeteners in surimi products The difference between this comparative example and Example 2 lies in the preparation of porous biochar, where the cyclodextrin derivative is replaced with cyclodextrin.

[0057] Experimental example: 1. Instruments and reagents Waters Xevo TQ-XS ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer (Waters Corporation, USA); Dionex ASE 350 accelerated solvent extraction system (Thermo Fisher Scientific, USA); AVANTIJ-E large-capacity high-speed refrigerated centrifuge (BEAKMAN Corporation, USA); BSA224S-CW electronic balance (accuracy 0.1 mg, Sartorius Scientific Instruments GmbH, Germany); Multi Reax multi-well vortex mixer (Hydorf GmbH, Germany); Milli-Q ultrapure water system (Milli-Q Corporation, USA).

[0058] All methanol was of chromatographic grade and purchased from Merck, Germany.

[0059] 2. Preparation of standard solutions Preparation of mixed standard solutions: Nine glycoside sweetener standards were separately prepared into 1.0 mg / L single standard stock solutions by adding them to methanol. The internal standard [U-13C]-stevioside was prepared into a 1.0 mg / L internal standard stock solution by adding it to methanol. Then, equal volumes of the nine single standard stock solutions were mixed and prepared into a series of intermediate mixed standard solutions with varying concentrations using methanol. An equal volume of internal standard stock solution was then added to each concentration of the intermediate mixed standard solution to obtain the mixed standard solution. The concentration gradients were 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL. In the preparation of the mixed standard solutions, the concentration of the internal standard [U-13C]-stevioside was 100 ng / mL.

[0060] 3. Chromatographic conditions in UPLC-MS / MS detection Chromatographic column: ACQUITY UPLC BEH C18 (2.1x100mm, 1.7μm); column temperature: 40℃; flow rate: 0.3mL / min; injection volume: 2μL; mobile phase: A is methanol, B is 5mmol / L ammonium acetate solution, and the specific gradient elution program is shown in Table 1.

[0061] Table 1 Mobile phase elution program for liquid chromatography

[0062] 4. Mass spectrometry conditions in UPLC-MS / MS detection The ion source was electrospray ionization (ESI); the scanning mode was negative ion scanning; and the acquisition mode was multiple reaction monitoring (MRM). Specific mass spectrometry parameters for 11 carbazoles and their halogenated derivatives are shown in Table 2.

[0063] Table 2. Mass spectrometry parameters of nine glycoside sweeteners

[0064] 5. Methodological Validation The prepared mixed standard solutions were analyzed by UPLC-MS / MS. The standard working curve was plotted with the peak area ratio as the ordinate (Y) and the mass concentration to internal standard concentration ratio as the abscissa (X, ng / ml). The linear range, linear regression equation, correlation coefficient, detection limit and quantitation limit of the nine glycoside sweeteners are shown in Table 3.

[0065] Table 3. Linear range, linear regression equation, and correlation coefficient of nine glycoside sweeteners.

[0066] 6. Spiked recovery test Fish surimi product samples were taken, and appropriate amounts of mixed standard working solutions of nine glycoside sweeteners were added to each sample. The spiking levels were 1, 3, and 10 times the limit of quantitation (LOQ) of each PHCZ. Six test solutions were prepared in parallel at each concentration level for analysis. The results are shown in Table 4. The average recoveries of the nine glycoside sweeteners ranged from 90.16% to 103.69%, and the relative standard deviations ranged from 2.24% to 7.22%. This method is stable, reliable, and has good accuracy and precision, meeting the requirements for the simultaneous detection of nine glycoside sweeteners in fish surimi products.

[0067] Table 4. Average recovery rates and relative standard deviations of nine glycoside sweeteners in surimi products.

[0068] In summary, the method for simultaneously detecting nine glycoside sweeteners in surimi products provided by this invention is stable, reliable, and has good accuracy and precision, meeting the needs for simultaneous detection of nine glycoside sweeteners in aquatic products.

[0069] The surface morphology of the porous biochar prepared in Example 2 was characterized by SEM, and the results are as follows: Figure 1 As shown, the porous biochar surface has wrinkles and pores, indicating that the porous biochar has a loose and porous structure.

[0070] In response to the high relative standard deviation of steviol disaccharide among the nine glycoside sweeteners at low spiking levels, this invention takes steviol disaccharide as an example. The average recovery rate and relative standard deviation of the samples after spiking at 5.0 µg / kg in the examples and comparative examples are shown in Table 5.

[0071] Table 5. Average recovery rates and relative standard deviations of nine glycoside sweeteners in surimi products.

[0072] This invention involves mixing surimi products with a methanol-water solution, centrifuging to remove the supernatant, purifying the supernatant with a purifying agent, and finally passing it through a membrane to obtain a surimi treatment solution. The surimi treatment solution is then analyzed using UPLC-MS / MS to detect the content of glycoside sweeteners. This invention uses isotopically labeled [U-13C]-stevioside as an internal standard to prepare a series of mixed standard solutions of nine glycoside sweeteners at various concentrations, and plots a standard curve that can be used to calculate the content of glycoside sweeteners in surimi products. The method of this invention has good detection effects on the nine glycoside sweeteners in surimi products. In the spiking method for detecting glycoside sweeteners, the lower the spiking amount, the greater the detection error. This application uses steviol disaccharide as an example to compare and analyze the detection effects of the embodiments and comparative examples of this invention. The porous biochar prepared in this invention can be used in the purifying agent to treat surimi products. In the preparation of the porous biochar, β-cyclodextrin and... p-Toluenesulfonyl cyclodextrin is prepared by reacting p-toluenesulfonyl chloride. Then, a cyclodextrin derivative is prepared by reacting p-toluenesulfonyl cyclodextrin with an amino compound, the amino compound being 2-aminopyrrole. The cyclodextrin derivative and chitosan are then mixed in a citric acid solution, the water is evaporated, and the mixture is calcined to prepare porous biochar. In this invention, the combined use of magnesium sulfate, PSA, and porous biochar demonstrates good detection efficacy for nine glycoside sweeteners in surimi products, with higher spiked recoveries and smaller relative standard deviations. Porous biochar exhibits good efficacy within a certain range of application; however, if the dosage is too low, the detection effect deteriorates. In preparing porous biochar, pentaerythritol tetracitrate can also be added. The pentaerythritol tetracitrate is mixed with the cyclodextrin derivative and chitosan in a citric acid solution to finally prepare porous biochar, which, when used in the treatment of surimi products, can improve the detection efficacy for the nine glycoside sweeteners in surimi products.

[0073] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0074] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for detecting nine glycoside sweeteners in surimi products, comprising: The surimi products were pretreated to obtain surimi treatment liquid, and nine glycoside sweeteners were detected in the surimi treatment liquid by UPLC-MS / MS. Purifying agents are used in the pretreatment process, including MgSO4, with the amount of MgSO4 used being 2-10 wt% of the surimi product.

2. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, It also includes the preparation of mixed standard solutions using the isotope internal standard method, and the construction of a standard working curve. The reagent for the isotope internal standard is [U-13C]-stevioside.

3. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The purifying agent also includes PSA, and the amount of PSA used is 0.3-2 wt% of the surimi product.

4. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The purifying agent also includes C18, and the amount of C18 used is 0.6-4 wt% of the surimi product.

5. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The purifying agent also includes porous biochar, and the amount of porous biochar used is 0.1-1 wt% of the surimi product.

6. The method for detecting nine glycoside sweeteners in surimi products according to claim 5, characterized in that, The porous biochar is prepared from cyclodextrin derivatives, chitosan, and citric acid. The cyclodextrin derivatives are formed by reacting p-toluenesulfonyl cyclodextrin with an amino compound, including 2-aminopyrrole.

7. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The pretreatment process first involves extraction with a methanol-water solution, followed by treatment with a purification agent, and finally membrane filtration.

8. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The amount of the surimi product used is 10-30 wt% of the methanol aqueous solution.

9. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The methanol content in the methanol-water solution is 10-60 vol.

10. The method for detecting nine glycoside sweeteners in surimi products according to claim 1, characterized in that, The nine glycoside sweeteners are steviol glycoside, ribobadiol A, ribobadiol B, ribobadiol C, ribobadiol D, ribobadiol F, durqueside A, steviol disaccharide, and stevia glycoside.

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