Pretreatment optimization method for measuring vitamin E in sea buckthorn seed oil

Abstract

The invention discloses a pretreatment optimization method for measuring vitamin E in sea buckthorn seed oil, and belongs to the technical field of analysis and detection. The invention aims to solve the problems of easy degradation of vitamin E, easy emulsification, low subsequent extraction efficiency and tedious operation in the saponification process of seabuckthorn seed oil in the prior art. The method comprises the following steps: firstly, mixing the sea buckthorn seed oil with an attapulgite and silicon dioxide composite adsorbent for pretreatment; under the protection of inert gas, a saponification reaction is carried out, and a saponification solution contains a composite antioxidant and a phase transfer catalyst; then carrying out automatic extraction by adopting a mixed solvent of ethyl acetate and methyl tert-butyl ether; and finally, purifying the extract through a solid-phase extraction column to obtain a vitamin E-containing component, and preparing the vitamin E-containing component into a test solution. The method is mainly used for providing an efficient, stable and reliable pretreatment sample for chromatographic determination of vitamin E in seabuckthorn seed oil. The invention discloses a pretreatment optimization method for measuring vitamin E in sea buckthorn seed oil.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology. More particularly, the present application relates to a pretreatment optimization method for determination of vitamin E in seabuckthorn seed oil. BACKGROUND

[0002] In the analysis of the nutritional components of seabuckthorn seed oil, accurate determination of the content of vitamin E is a key and challenging task, and the core difficulty lies in the sample pretreatment process. The existing conventional method usually contains saponification, extraction and purification steps, but when dealing with this special matrix of seabuckthorn seed oil, several interrelated technical bottlenecks are faced.

[0003] Vitamin E is sensitive to strong alkali, high temperature and oxygen, and is prone to degradation during saponification. However, in order to ensure complete hydrolysis of the oil, the traditional saponification process often needs to use a higher concentration of alkali, a longer reaction time or a higher temperature, which inevitably increases the risk of loss of the target substance. If the vitamin E is treated too gently to protect it, the saponification may not be complete, affecting subsequent analysis. In addition, the naturally occurring sterols, phospholipids and other components in seabuckthorn seed oil are extremely prone to serious emulsification under alkaline conditions, forming stable and difficult-to-demulsify emulsions, which leads to low efficiency and unstable recovery rate in the subsequent liquid-liquid extraction step.

[0004] In the extraction stage, the conventional method relies on multiple manual shaking and separation operations, which is time-consuming and increases the risk of sample oxidation and physical loss. The widely used extraction solvents such as n-hexane also have shortcomings in terms of toxicity and environmental friendliness. The crude extract obtained after extraction usually contains various coexisting interferents such as fatty acids and pigments, which will affect the accuracy of chromatographic analysis, and the conventional purification means often have problems such as long process, limited selectivity or poor reproducibility, making it difficult to maintain high purity while maintaining high recovery rate of the target substance.

[0005] Therefore, for the determination of vitamin E in seabuckthorn seed oil, the existing pretreatment technology still has obvious room for improvement in terms of how to balance the saponification efficiency and the stability of the target substance, effectively prevent emulsification, achieve safe and efficient extraction and specific purification, and simplify the overall operation process. SUMMARY

[0006] It is an object of the present application to solve at least the above problems and to provide at least the advantages to be explained later.

[0007] It is another object of the present application to provide a pretreatment optimization method for determination of vitamin E in seabuckthorn seed oil, which aims to solve the technical problems of existing methods that saponification process easily leads to degradation and emulsification of vitamin E, low extraction efficiency and complicated operation, and complex and long overall pretreatment process leading to poor accuracy and stability of determination results.

[0008] To achieve these objects and other advantages of the present application, a pretreatment optimization method for vitamin E determination in seabuckthorn seed oil is provided, which comprises the following steps: Step one, mix seabuckthorn seed oil with adsorbent at a mass ratio of 2-1:1, stir at 40-50℃ for 20-40min, then remove the adsorbent by centrifugation to obtain pretreated seabuckthorn seed oil; the adsorbent comprises attapulgite and silicon dioxide at a mass ratio of 3-4:1; Step two, mix the pretreated seabuckthorn seed oil with saponification solution at a mass to volume ratio of 1:15-25 under inert gas protection, oscillate in a 45-55℃ water bath for 5-10min to obtain saponified product; add sodium chloride with a mass concentration of 2-4% to the saponification system during the saponification reaction; the saponification solution is composed of the following components: potassium hydroxide ethanol solution with a concentration of 0.8-1.2mol / L as the reaction matrix, and contains 0.2-0.4% w / v of L-ascorbic acid, 0.06-0.08% w / v of propyl gallate as a composite antioxidant, and 0.02-0.04mol / L of tetrabutylammonium bromide as a phase transfer catalyst; Step three, add organic solvent to the saponified product, perform extraction in a closed automated extraction device, collect the extract and concentrate to remove the solvent to obtain the extract; Step four, purify the extract using a solid phase extraction column, sequentially elute with n-hexane and a mixed solution of acetonitrile-dichloromethane at a volume ratio of 1:1 to collect the vitamin E-containing component; Step five, concentrate the collected vitamin E-containing component to remove the solvent under nitrogen flow, then redissolve with n-hexane or methanol and make up to volume to obtain the test solution for vitamin E chromatographic analysis.

[0009] Preferably, in the saponification reaction of step two, an energy gradient ultrasonic treatment is applied simultaneously, specifically: within 0-2min of the start of the saponification reaction, a high-frequency low-power mode is used, with a frequency of 80kHz and a power of 50-80W; during the subsequent 3-8min main reaction period, switch to a low-frequency high-power mode, with a frequency of 20kHz and a power of 150-200W.

[0010] Preferably, the preparation method of the adsorbent for the pretreatment optimization method for vitamin E determination in seabuckthorn seed oil comprises the following steps: 1) disperse attapulgite in deionized water, add hexadecyltrimethylammonium bromide equivalent to 5-10% of the mass of attapulgite, stir at 70-80℃ for 2-4h to obtain organic attapulgite; 2) Disperse organic attapulgite and silica in an ethanol-water mixture at a mass ratio of 3-4:1, add 1-3% of 3-aminopropyltriethoxysilane by mass of silica, and react at 60-70℃ for 4-6 hours to form a composite material with attapulgite as the core and silica as the shell. 3) The composite material obtained in step 2) is immersed in a 0.5-1.0 mol / L citric acid-sodium citrate buffer solution and activated at 40-50℃ for 3-5 h. Then it is filtered, washed, and calcined at 250-300℃ for 1-2 h to obtain the final product.

[0011] Preferably, in the pretreatment optimization method for determining vitamin E in sea buckthorn seed oil, the packing material of the solid-phase extraction column in step four is silica microspheres with a vitamin E molecularly imprinted polymer layer grafted onto their surface. The preparation method specifically includes the following steps: a) Disperse 1 part by weight of silica microspheres in toluene, add 10-15% by weight of vinyltriethoxysilane, and heat under reflux at 80-85°C for 6-8 hours; after the reaction is completed, wash and dry to obtain silica microspheres with vinyl-modified surface. b) Using δ-tocopherol as a template molecule, it is dissolved with the functional monomer 4-vinylpyridine and the crosslinking agent ethylene glycol dimethacrylate in a molar ratio of 1:4:20 in a second solvent containing a porogen-acetonitrile mixture to form a prepolymerization solution; in the second solvent, the volume ratio of acetonitrile to toluene is 1:1-2; in the prepolymerization solution, the total mass of the template molecule, functional monomer, and crosslinking agent accounts for 15-25% of the volume of the second solvent; c) Add the vinyl-modified silica microspheres obtained in step a) to the prepolymerization solution prepared in step b), purge with nitrogen to remove oxygen, and add azobisisobutyronitrile (azoIOB) initiator at 1% of the mass of the crosslinking agent. Heat-initiate the polymerization reaction at 60-65°C for 12-16 hours to obtain the grafted polymer. d) The grafted polymer was continuously extracted for 24-48 hours using a methanol-acetic acid solution with a volume ratio of 9:1 by Soxhlet extraction.

[0012] Preferably, in the pretreatment optimization method for determining vitamin E in sea buckthorn seed oil, step two of the saponification solution further contains 0.01-0.02 mol / L of disodium ethylenediaminetetraacetate as a metal chelating agent.

[0013] Preferably, in the pretreatment optimization method for determining vitamin E in sea buckthorn seed oil, in step three, the automated extraction device synchronously applies vortex oscillation during the extraction process, with an oscillation frequency of 1000-1500 rpm and an oscillation time of 3-5 min.

[0014] Preferably, in the pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil, in step four, the solid phase extraction column is first equilibrated with n-hexane before elution, with an equilibration volume of 5-10 mL.

[0015] Preferably, in the pretreatment optimization method for determining vitamin E in sea buckthorn seed oil, during the energy gradient ultrasonic treatment, a gradual transition period of 30-60 seconds is provided when switching frequencies, during which the frequency linearly decreases from 80kHz to 20kHz and the power linearly increases from 80W to 150W.

[0016] Preferably, in the pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil, before using the adsorbent in step one, the adsorbent is vacuum activated at 150-200℃ for 1-2 hours, and then stored in a dry inert atmosphere for later use.

[0017] The present invention has at least the following beneficial effects: 1. To address the challenge of vitamin E's easy degradation under strong alkali and high temperature conditions, this invention proposes saponification under inert gas protection, introducing a complex antioxidant (L-ascorbic acid, propyl gallate) and a metal chelating agent (disodium ethylenediaminetetraacetate). This technical solution effectively isolates oxygen, inhibits free radical chain reactions, and complexes the metal ions involved in catalytic degradation. Thus, under mild saponification conditions (45–55℃, 5–10 min), complete hydrolysis of the fat is achieved while maximizing the preservation of vitamin E's chemical integrity, ensuring the accuracy and reliability of subsequent chromatographic analysis.

[0018] 2. To address the problem of severe emulsification during the saponification process of sea buckthorn seed oil, which leads to extraction difficulties, this invention employs a composite adsorbent of attapulgite and silica for oil sample pretreatment, and adds sodium chloride to the saponification system. This pretreatment adsorbs natural emulsifying components (such as phospholipids and sterols), while the addition of sodium chloride disrupts the emulsion layer structure through salting-out, significantly reducing the degree of emulsification. This results in a clear liquid-liquid extraction interface, rapid separation, and a substantial improvement in vitamin E recovery rate and operational reproducibility.

[0019] 3. To address the problems of cumbersome traditional manual extraction operations, high solvent toxicity, and easy oxidation loss, this invention employs an ethyl acetate-methyl tert-butyl ether mixed solvent system and combines it with vortex oscillation in an automated extraction device. This mixed solvent has moderate polarity, exhibits good extraction capabilities for various vitamin E homologues, and is low in toxicity and environmentally friendly. Vortex oscillation enhances the mass transfer process, enabling efficient extraction to be completed within 3–5 minutes in a closed system, thus shortening pretreatment time and reducing human error and the risk of sample oxidation.

[0020] 4. To address the issues of numerous interfering substances in crude extracts and lengthy purification steps, this invention employs silica microspheres with a surface-grafted vitamin E molecularly imprinted polymer (MIP) as a solid-phase extraction packing material. This MIP material exhibits high selectivity for vitamin E, specifically adsorbing the target analyte and effectively removing interfering components such as fatty acids and pigments. Combined with column equilibration, rapid and efficient sample purification is achieved, significantly improving the sensitivity and specificity of chromatographic analysis while reducing solvent usage and operational steps, making it suitable for high-throughput, standardized detection scenarios.

[0021] 5. This invention further integrates energy gradient ultrasound-assisted saponification, surface-modified composite adsorbents, and their activation processes. Different frequency-power combinations are used in different reaction stages of the ultrasonic treatment, which both promotes the saponification reaction rate and avoids localized overheating that could destroy vitamin E. After organic-inorganic composite modification and high-temperature activation, the adsorbent's adsorption capacity and selectivity are significantly improved. These synergistic measures achieve an optimal balance between efficiency, protection, and purification in the entire pretreatment process, making it suitable for the stable and high-precision determination of trace amounts of vitamin E in complex oil matrices.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0024] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0025] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0026] Example 1 This invention provides a pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil, which includes the following steps: Step 1, Oil Sample Pretreatment: Weigh 2.0 g of sea buckthorn seed oil sample into a 50 mL stoppered centrifuge tube, add 1.0 g of pretreatment adsorbent, and place in a 45℃ constant temperature water bath. Stir mechanically at 300 rpm for 30 minutes. After stirring, centrifuge at 5000 rpm for 15 minutes. Carefully transfer the clear upper oil sample, avoiding aspiration of the bottom adsorbent, to obtain the pretreated sea buckthorn seed oil. The adsorbent is a physical mixture of attapulgite and fumed silica at a mass ratio of 3.5:1.

[0027] Step 2, Saponification reaction under inert gas protection: In a glove box filled with high-purity nitrogen (purity >99.999%), accurately weigh 1.00 g of pretreated sea buckthorn seed oil obtained in step 1 and place it in a 30 mL alkali-resistant reaction flask. Add 20 mL (i.e., a mass-to-volume ratio of 1:20) of freshly prepared saponification solution to the reaction flask. Add solid sodium chloride to the reaction system so that its mass concentration in the saponification solution is approximately 3%. The composition of the saponification solution is as follows: reaction matrix: 1.0 mol / L potassium hydroxide in anhydrous ethanol solution. Composite antioxidants: L-ascorbic acid (0.3% w / v), propyl gallate (0.07% w / v). Phase transfer catalyst: tetrabutylammonium bromide (0.03 mol / L). Seal the reaction flask and continuously purge with nitrogen as a headspace protective gas. Place the reaction flask in a 50°C constant temperature water bath shaker and shake at 150 rpm for 8 minutes. After the reaction is complete, a clear saponification solution without visible emulsion is obtained.

[0028] Step 3, Automated Mixed Solvent Extraction: Transfer all the saponified liquid obtained in Step 2 to the extraction chamber of a fully automated parallel concentration extractor (model: APLE-1000). Add 20 mL of an organic solvent consisting of ethyl acetate and methyl tert-butyl ether in a 1:1 (v / v) ratio.

[0029] Start the vortex extraction program on the instrument: set the oscillation frequency to 1200 rpm and the oscillation time to 4 minutes. After the program ends, let it stand for 2 minutes until the interface between the two phases is clear. The instrument automatically collects the upper organic phase. Repeat the extraction once more with 10 mL of mixed solvent, and combine the two organic extracts. Transfer the combined extract to a concentration tube and concentrate it to near dryness (approximately 0.5 mL) under a vacuum of -0.09 MPa in a 40°C water bath, obtaining a viscous extract.

[0030] Step 4: Solid-phase extraction (SPE) purification: SPE column preparation: Take a 500 mg / 6 mL aminopropyl silica gel (NH2) solid phase extraction column (silica gel column can also be used), and first equilibrate the column with 5 mL of n-hexane at a flow rate of about 1 mL / min.

[0031] Sample loading: Dissolve the extract obtained in step 3 in 2 mL of n-hexane and load the entire sample onto a pre-equilibrated SPE column.

[0032] Elution and elution: Elute sequentially with 5 mL of n-hexane, discarding the eluent (mainly to remove nonpolar impurities). Then elute with 5 mL of a solution of acetonitrile and dichloromethane in a 1:1 (v / v) ratio. Collect this eluent in a glass test tube; this fraction contains vitamin E.

[0033] Step 5, Preparation of test solution: The vitamin E-containing component collected in step 4 was concentrated by gentle nitrogen purging in a 40°C water bath until completely dry.

[0034] Dissolve the residue in 1.00 mL of chromatographically pure hexane and vortex for 30 seconds to ensure complete dissolution.

[0035] The solution was filtered through a 0.22 μm polytetrafluoroethylene (PTFE) organic phase needle filter, and the filtrate was transferred to a 2 mL chromatographic injection bottle to obtain the test solution for vitamin E determination.

[0036] Example 2 The difference between this embodiment and Embodiment 1 is that energy gradient ultrasound-assisted technology is introduced, while the other parameters and process steps are the same as in Embodiment 1.

[0037] Modify the procedure for step 2 (saponification): Place the reaction flask in a programmable ultrasonic cleaning tank (with temperature control) for the saponification reaction. The ultrasonic program is set as follows: 0-2 minutes (initial phase): frequency 80kHz, power 60W, continuous mode. 2 minutes 00 seconds - 2 minutes 45 seconds (gradual transition phase): frequency linearly decreases from 80kHz to 20kHz, power linearly increases from 60W to 150W. 2 minutes 45 seconds - 8 minutes (main reaction phase): frequency 20kHz, power 180W, pulse mode (on for 2 seconds, off for 1 second). The water bath temperature remains controlled at 50℃, and the total reaction time is 8 minutes. This process is carried out under continuous nitrogen bubbling protection.

[0038] Example 3 The difference between this embodiment and Embodiment 1 is that a composite adsorbent is used instead of the physically mixed adsorbent in Embodiment 1, while the remaining parameters and process steps are the same as in Embodiment 1.

[0039] Preparation and activation of composite adsorbent: Organic attapulgite: Weigh 10.0 g of attapulgite and disperse it in 200 mL of deionized water. Add 0.75 g (7.5 wt%) of hexadecyltrimethylammonium bromide (CTAB) and stir vigorously at 75 °C for 3 hours. After the reaction, filter and wash alternately with deionized water and ethanol until no Br is visible. - (AgNO3 detection), dried at 80℃, and ground through a 200-mesh sieve.

[0040] Synthesis of core-shell composite material: The above-mentioned organic attapulgite clay and fumed silica were mixed at a mass ratio of 3.5:1 and dispersed in 150 mL of ethanol-water (4:1, v / v) solution. 2% (by mass of silica) of 3-aminopropyltriethoxysilane (APTES) was added, and the mixture was refluxed and stirred at 65 °C for 5 hours. After cooling, the mixture was filtered, washed with ethanol, and dried.

[0041] Acid activation and calcination: The composite material was impregnated in 0.8 mol / L citrate-sodium citrate buffer (pH 4.5) and activated by gentle shaking at 45°C for 4 hours. After filtration, it was washed with deionized water until neutral, dried in an oven at 120°C, and then transferred to a muffle furnace for calcination at 280°C for 1.5 hours.

[0042] Vacuum activation and storage: The calcined adsorbent was transferred to a vacuum drying oven and activated under vacuum at 180℃ and -0.1MPa for 2 hours. It was then transferred to a desiccator under nitrogen atmosphere protection and used within one week.

[0043] Using the self-made adsorbent described above, the operation was carried out exactly in accordance with step 1 (pretreatment) and all subsequent steps of Example 1.

[0044] Example 4 The difference between this embodiment and Embodiment 1 is that a self-made molecularly imprinted polymer (MIP) solid-phase extraction column is used for purification in step 4, while the other parameters and process steps are the same as in Embodiment 1.

[0045] Specifically, in step four, the packing material for the solid-phase extraction column is silica microspheres with a vitamin E molecularly imprinted polymer layer grafted onto their surface. The preparation method includes the following steps: Preparation of MIP-SPE columns: Vinylation of silica gel surface: 5.0 g of silica gel microspheres (50 μm particle size, 100 Å pore size) were weighed and dispersed in 100 mL of anhydrous toluene. 0.6 g of vinyltriethoxysilane (VTES) was added, and the mixture was refluxed at 83 °C under nitrogen protection for 7 hours. After the reaction, the mixture was washed successively with toluene and acetone, and dried under vacuum at 60 °C to obtain vinyl-modified silica gel (Vi-SiO2).

[0046] Preparation of prepolymerization solution: Using δ-tocopherol as the template molecule, weigh it together with the functional monomer 4-vinylpyridine and the crosslinking agent ethylene glycol dimethacrylate in a molar ratio of 1:4:20. Dissolve it in 40 mL of acetonitrile-toluene (1:1.5, v / v) mixed solvent, and sonicate to form a homogeneous prepolymerization solution.

[0047] Surface-imprinted polymerization: 2.0 g Vi-SiO2 was added to the above prepolymer solution, and nitrogen gas was purged for deoxygenation for 30 minutes. Azobisisobutyronitrile (AIBN, 1% by mass of the crosslinking agent) was added as the initiator, and thermal polymerization was initiated in an oil bath at 62°C for 16 hours. After the reaction was complete, the mixture was filtered and thoroughly washed with acetonitrile.

[0048] Template molecule elution: The obtained solid was loaded into a Soxhlet extractor and extracted continuously for 36 hours using methanol:acetic acid (9:1, v / v) as the extraction solvent. It was then washed with pure methanol until neutral and dried under vacuum at 60°C to obtain the MIP-SPE packing material.

[0049] Column packing: Dry pack 200mg MIP packing material into an empty 3mL SPE column tube, and compact it by adding sieve plates at the top and bottom.

[0050] Application: In step 4 (purification) of Example 1, the self-made MIP-SPE column described above is used. The procedure is as follows: Equilibration: Equilibrate with 5 mL of n-hexane.

[0051] Sample loading: Dissolve the extract in n-hexane and load the sample.

[0052] Rinse: Rinse with 5 mL of n-hexane and discard.

[0053] Elution: Elute with 5 mL of acetonitrile-dichloromethane (1:1, v / v) and collect the fraction for subsequent analysis.

[0054] Example 5 The difference between this embodiment and Embodiment 1 is that a metal chelating agent is added to the saponification solution, while the other parameters and process steps are the same as in Embodiment 1.

[0055] Specifically, when preparing the saponification solution, in addition to the components in Example 1, disodium ethylenediaminetetraacetate (EDTA-2Na) is added to make its concentration in the saponification solution 0.015 mol / L.

[0056] Example 6 The difference between this embodiment and Embodiment 1 is that the extraction and SPE equilibrium parameters are defined in more detail, while the other parameters and process steps are the same as in Embodiment 1.

[0057] Specifically, in step three, the vortex oscillation frequency in the automated extraction device is set to 1500 rpm, and the oscillation time is 3 minutes. Other aspects are the same as in Example 1.

[0058] In step four, before use, the solid-phase extraction column is fully equilibrated with 10 mL of n-hexane at a flow rate of approximately 2 mL / min to ensure that the column bed is completely wetted and activated.

[0059] Comparative Example 1: The classic saponification extraction method for vegetable oil determination in GB5009.82-2016 (refluxing saponification at 80℃ for 30 minutes, without inert gas protection, manual n-hexane extraction).

[0060] Comparative Example 2: Saponification was performed only (under the same conditions as Example 1), but the adsorbent pretreatment in step 1 was omitted, and sodium chloride was not added to the saponification solution.

[0061] Comparative Example 3: Hexane was used as the extraction solvent, and extraction was carried out by manual shaking and separation by a separatory funnel. Other steps were the same as in Example 1.

[0062] Comparative Example 4: Purification was performed using a standard C18 solid-phase extraction column, with methanol as the elution solvent. Other steps were the same as in Example 1.

[0063] Experimental Examples and Effect Verification 1. Testing Methods and Indicators Instrument: High performance liquid chromatography-fluorescence detector (HPLC-FLD).

[0064] Chromatographic conditions: C18 column (250 × 4.6 mm, 5 μm); mobile phase: methanol; flow rate: 1.0 mL / min; column temperature: 30℃; fluorescence detection: λ ex =294nm,λ em =326nm.

[0065] Evaluation indicators: Overall recovery rate: Vitamin E mixed standards (α-, β-, γ-, δ-tocopherol) were added to sea buckthorn seed oil with known low background, and the recovery rate of each component was determined after pretreatment. The arithmetic mean of the recovery rates of each component was calculated.

[0066] Precision: Six parallel oil samples were processed, and the relative standard deviation (RSD) of the total vitamin E content determination was calculated.

[0067] Anti-emulsification effect: After saponification, let stand for 5 minutes, observe and record the degree of emulsification (1-clear, 2-slightly cloudy, 3-emulsified, 4-severely emulsified).

[0068] Chromatogram quality: Observe the target peak shape, resolution (Rs>1.5) and baseline interference.

[0069] Pretreatment time: The total operation time from weighing the sample to obtaining the test solution.

[0070] 2. The test results are shown in Table 1.

[0071] Table 1 Test Results

[0072] As can be seen from the comparison of data in Table 1: Example 1 (recovery rate 95.8%, RSD 1.9%) was significantly better than Comparative Example 1 (recovery rate 72.4%, RSD 6.8%), demonstrating that the core combination scheme of the present invention, namely "adsorption pretreatment + antiemulsifying salt + inert protection + composite antioxidant", effectively overcomes the problems of severe vitamin E degradation, difficulty in breaking emulsions, and large fluctuations in results caused by the traditional high-temperature saponification method.

[0073] Example 1 (95.8% recovery, no emulsification) was significantly better than Comparative Example 2 (81.5% recovery, severe emulsification), confirming that "adsorption pretreatment" and "addition of sodium chloride to the saponification solution" are key steps to prevent system emulsification and ensure the smooth progress of liquid-liquid extraction.

[0074] Example 2 (recovery rate 97.5%) was slightly higher than Example 1 (95.8%), indicating that "energy gradient ultrasound assistance" further enhanced the saponification mass transfer process and improved the reaction efficiency under mild conditions.

[0075] Example 4 (using a MIP-SPE column, recovery rate 98.2%, RSD 1.2%) is superior to Comparative Example 4 (using a conventional C18 column, recovery rate 92.1%, RSD 2.9%) in both recovery rate and precision, demonstrating that the "Vitamin E molecularly imprinted polymer solid-phase extraction column" has significant specific recognition and purification capabilities, effectively removing impurities and improving detection accuracy.

[0076] In the storage stability test, Example 5 (with added EDTA-2Na) showed the smallest decrease in recovery rate, indicating that the "metal chelating agent" can enhance the stability of the test solution by integrating metal ions to inhibit oxidative degradation during storage.

[0077] In Example 3, after using the modified adsorbent, the chromatographic baseline was cleaner than that in Example 1, reflecting that the "modified composite adsorbent" has a selective adsorption effect on coexisting impurities such as pigments and phospholipids, which helps to improve the chromatographic separation effect.

[0078] The pretreatment time of Example 6 (55 minutes) was slightly shorter than that of Example 1 (60 minutes), demonstrating that by optimizing the extraction oscillation intensity and the equilibrium volume of the SPE column, operational efficiency can be further improved while maintaining high-efficiency recovery.

[0079] In summary, the optimized pretreatment method for the determination of vitamin E in sea buckthorn seed oil provided by this invention systematically solves the problems of easy degradation, easy emulsification, low extraction and purification efficiency, and cumbersome operation in the pretreatment of vitamin E determination in sea buckthorn seed oil through a series of synergistic technical means. The preferred technical features can be flexibly combined according to actual detection needs, further providing gains in selectivity, stability, or operational efficiency while ensuring high recovery rate and high precision. This provides a reliable pretreatment solution for the accurate, rapid, and stable determination of vitamin E in complex oil samples.

[0080] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0081] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. An optimized pretreatment method for the determination of vitamin E in sea buckthorn seed oil, characterized in that, Includes the following steps: Step 1: Mix sea buckthorn seed oil and adsorbent at a mass ratio of 2-1:1, stir at 40-50℃ for 20-40 minutes, then centrifuge to remove the adsorbent to obtain pretreated sea buckthorn seed oil; the adsorbent includes attapulgite and silica at a mass ratio of 3-4:

1. Step 2: Under inert gas protection, mix the pretreated sea buckthorn seed oil with the saponification solution at a mass-to-volume ratio of 1:15-25, and shake the mixture in a water bath at 45-55℃ for 5-10 minutes to obtain the saponified product; during the saponification reaction, add sodium chloride with a mass concentration of 2-4% to the saponification system. The saponification solution consists of the following components: a potassium hydroxide ethanol solution with a concentration of 0.8-1.2 mol / L as the reaction matrix, containing 0.2-0.4% w / v L-ascorbic acid, 0.06-0.08% w / v propyl gallate as a composite antioxidant, and 0.02-0.04 mol / L tetrabutylammonium bromide as a phase transfer catalyst; Step 3: Add organic solvent to the saponified product, extract in a closed automated extraction device, collect the extract and concentrate to remove the solvent to obtain the extract; the organic solvent is a mixture of ethyl acetate and methyl tert-butyl ether in a volume ratio of 1:

1. Step 4: Purify the extract using a solid-phase extraction column, eluting sequentially with a mixture of n-hexane and acetonitrile-dichloromethane in a 1:1 volume ratio, and collect the component containing vitamin E. Step 5: The collected vitamin E-containing components are purged and concentrated under a nitrogen stream until the solvent is removed. Then, they are redissolved in hexane or methanol and brought to a final volume to obtain a test solution for vitamin E chromatographic analysis.

2. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 1, characterized in that, During the saponification reaction in step two, an energy gradient ultrasonic treatment is applied simultaneously. Specifically, during the initial 0-2 minutes of the saponification reaction, a high-frequency low-power mode is used, with a frequency of 80kHz and a power of 50-80W. During the subsequent 3-8 minutes of the main reaction period, the mode is switched to a low-frequency high-power mode, with a frequency of 20kHz and a power of 150-200W.

3. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 1, characterized in that, The preparation method of the adsorbent includes the following steps: 1) Disperse attapulgite in deionized water, add 5-10% of cetyltrimethylammonium bromide by weight of attapulgite, and stir at 70-80℃ for 2-4 hours to modify it, thus obtaining organic attapulgite. 2) Disperse organic attapulgite and silica in an ethanol-water mixture at a mass ratio of 3-4:1, add 1-3% of 3-aminopropyltriethoxysilane by mass of silica, and react at 60-70℃ for 4-6 hours to form a composite material with attapulgite as the core and silica as the shell. 3) The composite material obtained in step 2) is immersed in a 0.5-1.0 mol / L citric acid-sodium citrate buffer solution and activated at 40-50℃ for 3-5 h. Then it is filtered, washed, and calcined at 250-300℃ for 1-2 h to obtain the final product.

4. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 1, characterized in that, In step four, the packing material for the solid-phase extraction column is silica microspheres with a vitamin E molecularly imprinted polymer layer grafted onto their surface. The preparation method specifically includes the following steps: a) Disperse 1 part by weight of silica microspheres in toluene, add 10-15% by weight of vinyltriethoxysilane, and heat and reflux at 80-85℃ for 6-8 hours; after the reaction is completed, wash and dry to obtain silica microspheres with vinyl-modified surface. b) Using δ-tocopherol as a template molecule, it is dissolved with the functional monomer 4-vinylpyridine and the crosslinking agent ethylene glycol dimethacrylate in a molar ratio of 1:4:20 in a second solvent containing a porogen-acetonitrile mixture to form a prepolymerization solution; in the second solvent, the volume ratio of acetonitrile to toluene is 1:1-2; in the prepolymerization solution, the total mass of the template molecule, functional monomer, and crosslinking agent accounts for 15-25% of the volume of the second solvent; c) Add the vinyl-modified silica microspheres obtained in step a) to the prepolymerization solution prepared in step b), purge with nitrogen to remove oxygen, and add azobisisobutyronitrile (azoIOB) initiator at 1% of the mass of the crosslinking agent. Heat-initiate the polymerization reaction at 60-65°C for 12-16 hours to obtain the grafted polymer. d) The grafted polymer was continuously extracted for 24-48 hours using a methanol-acetic acid solution with a volume ratio of 9:1 by Soxhlet extraction.

5. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 1, characterized in that, In step two, the saponification solution also contains 0.01-0.02 mol / L of disodium ethylenediaminetetraacetate as a metal chelating agent.

6. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 1, characterized in that, In step three, the automated extraction device applies vortex oscillation synchronously during the extraction process, with an oscillation frequency of 1000-1500 rpm and an oscillation time of 3-5 min.

7. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 1, characterized in that, In step four, before elution, the solid-phase extraction column is equilibrated with n-hexane, with an equilibration volume of 5-10 mL.

8. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 2, characterized in that, In energy gradient ultrasound processing, a gradual transition period of 30-60 seconds is set when switching frequencies, during which the frequency linearly decreases from 80kHz to 20kHz and the power linearly increases from 80W to 150W.

9. The pretreatment optimization method for the determination of vitamin E in sea buckthorn seed oil as described in claim 3, characterized in that, Before using the adsorbent in step one, the adsorbent is vacuum activated at 150-200℃ for 1-2 hours, and then stored in a dry inert atmosphere for later use.

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