Method for detecting vitamin E in vegetables

Abstract

The invention discloses a method for detecting vitamin E in vegetables, and belongs to the technical field of medicine analysis and detection.The method comprises the steps that isopropyl alcohol is added into vegetable plant tissue dry powder, dissolution, vortex oscillation, standing in a dark place at the temperature of 4 DEG C, normal-temperature extraction is conducted in an ultrasonic cleaner, then centrifugation is conducted, supernate is sucked, and the supernate is blow-dried through a nitrogen blowing instrument; the method comprises the following steps: dissolving a sample in a chromatographic column, dissolving the sample in chromatographically pure n-hexane, standing, passing through an organic filter membrane, and detecting on a machine, adopting a normal-phase chromatographic column Hypersil GOLD Silica, taking the ratio of n-hexane to tert-butyl methyl ether being 94: 6 as a mobile phase, and controlling the column temperature at 25 DEG C, the flow velocity at 0.6 mL / min, the sample size at 6 mu L and the ultraviolet detection wavelength at 295 nm. According to the method for detecting the vitamin E in the vegetables, provided by the invention, a small amount of sample is needed during detection, only 0.2 g of dried sample powder is needed, the detection of 8 kinds of VE isomeride can be completed by adopting a high performance liquid chromatograph (normal phase), and the detection efficiency is high.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis and detection, and particularly relates to a method for detecting vitamin E in vegetables. Background Art

[0002] Vitamin E (VE, Tocopherol) is also known as tocopherol. Naturally occurring VE can be divided into 8 isomers according to the number and position of methyl groups on its chromophore, including 4 tocopherols (α-tocopherol - α-T, β-tocopherol - β-T, γ-tocopherol - γ-T, δ-tocopherol - δ-T) and 4 unsaturated forms of tocotrienols (α-tocotrienol - α-3T, β-tocotrienol - β-3T, γ-tocotrienol - γ-3T, δ-tocotrienol - δ-3T), among which α-tocopherol (α-T) has the highest biological activity.

[0003] The detection methods of VE mainly include colorimetry, ultraviolet spectrophotometry, infrared spectroscopy, fluorescence method, gas chromatography, liquid chromatography, etc. Among them, the detection limits of colorimetry, ultraviolet spectrophotometry and infrared spectroscopy are relatively low and the sensitivity is poor; while the instrument accessories of fluorescence method and gas chromatography are relatively expensive and difficult to popularize in ordinary laboratories. High performance liquid chromatography is widely used by most testing institutions and universities due to its advantages such as high sensitivity, good reproducibility, high stability and high laboratory popularity.

[0004] At present, the content of VE isomers is usually determined by the national standard methods GB 5009.82 - 2016 "National Food Safety Standard Determination of Vitamins A, D, E in Foods" and GB / T 26635 - 2011 "Determination of Tocopherols and Tocotrienols in Animal and Vegetable Oils - High Performance Liquid Chromatography Method". Among them, "National Food Safety Standard Determination of Vitamins A, D, E in Foods" includes two methods, reverse-phase liquid chromatography and normal-phase liquid chromatography, to determine the content of VE isomers, but both methods can only determine α-T, β-T, γ-T, δ-T in edible oils and do not mention the detection of α-3T, β-3T, γ-3T, δ-3T. The method described in "Determination of Tocopherols and Tocotrienols in Animal and Vegetable Oils - High Performance Liquid Chromatography Method" can simultaneously determine 8 isomers such as α-T, β-T, γ-T, δ-T and α-3T, β-3T, γ-3T, δ-3T in edible oils, which is an important method reference for simultaneously detecting 8 VE isomers; the more VE isomers are detected, the higher the requirements for the chromatographic column; for the 8 isomers of VE, this standard recommends two chromatographic columns for separation, a diol-based silica gel column, which can effectively separate the 8 isomers, but this chromatographic column is expensive and has a low popularity in laboratories, and the silica gel column cannot separate β-3T and γ-T, causing certain difficulties in accurately determining the content of each isomer.

[0005] In the prior art, a high performance liquid chromatography-based fluorescence analysis method for 8 VE isomers in Fritillaria thunbergii Miq. flowers and pollen is disclosed. In this method, the sample amount required for VE extraction is large, and the extraction solvent is n-hexane, which is volatile and has obvious toxicity. When using normal phase high performance liquid chromatography, the detection process takes a long time. The lower limit and the upper limit of the linear detection range of the obtained linear regression equation only have a 10-fold difference, and the detection range is narrow. Summary of the Invention

[0006] In view of the above problems, the present invention provides a method for detecting vitamin E in vegetables, effectively solving the technical problems of long detection time and narrow detection range caused by using normal phase high performance liquid chromatography in the prior art. At the same time, a method capable of simultaneously detecting 8 VE isomers in vegetables is provided, with short detection time and wide detection range.

[0007] The present invention provides a method for detecting vitamin E in vegetables, comprising the following steps:

[0008] Prepare single standard working solutions, and respectively obtain α-Tocopherol single standard working solution, β-Tocopherol single standard working solution, γ-Tocopherol single standard working solution, δ-Tocopherol single standard working solution, α-3T Tocotrienol single standard working solution, β-3T Tocotrienol single standard working solution, γ-3T Tocotrienol single standard working solution, and δ-3T Tocotrienol single standard working solution, each with a concentration of 128 μg / mL. Perform high performance liquid chromatography analysis and record the retention times of the chromatographic peaks of 8 vitamin E isomers respectively.

[0009] Mix the single standard stock solutions of 8 vitamin E isomers, add n-hexane, and prepare a mixed standard working solution with a concentration of 128 μg / mL. Dilute it with n-hexane to obtain mixed standard solutions with concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL respectively. Perform high performance liquid chromatography analysis on the mixed standard solutions, and determine the corresponding relationship between the chromatographic peaks of the mixed standard solutions and 8 vitamin E isomers according to the retention times of the chromatographic peaks, and draw the standard curves of 8 vitamin E isomers respectively.

[0010] Treatment of the sample to be measured: Place the vegetable plant tissue in a forced-air drying oven at 60 °C and dry it to a constant weight. Grind it into powder using an ultra-high-throughput grinder, place it in a -40 °C environment, store it in the dark and sealed for later use. Add isopropanol to the dried vegetable plant tissue powder, invert it up and down until the solid is completely dissolved. After vortexing for 30 s, place it in a 4 °C environment, let it stand in the dark for 1 h, perform ultrasonic extraction and centrifugation to obtain the supernatant. Aspirate the supernatant and dry it with a nitrogen blower. Redissolve it in chromatographically pure n-hexane, let it stand and filter to obtain the sample solution to be measured.

[0011] Sample injection and detection: Inject the sample solution to be measured into a special injection vial for liquid chromatography. Use high-performance liquid chromatography to detect the peak areas of 8 vitamin E isomers in the sample solution to be measured, and calculate the contents of the 8 vitamin E isomers by corresponding to the standard curves of the 8 vitamin E isomers. During detection, the liquid chromatography column is Hypersil GOLDSilica, with a specification of 250×4.6 mm, 5 μm; the mobile phase is a composition of n-hexane and tert-butyl methyl ether with a volume ratio of 94:6, the column temperature of the chromatographic column is 20 °C - 30 °C, the flow rate is 0.4 mL / min - 0.8 mL / min, and the injection volume is 6 μL - 10 μL.

[0012] As a preferred embodiment, the detection temperature of the liquid chromatography column is 25 °C.

[0013] As a preferred embodiment, the flow rate of the mobile phase is 0.6 mL / min.

[0014] As a preferred embodiment, the injection volume is 6 μL.

[0015] As a preferred embodiment, the liquid chromatography is detected using an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 295 nm.

[0016] As a preferred embodiment, the detection time is 20 min - 25 min.

[0017] As a preferred embodiment, the dosage ratio of the dried vegetable plant tissue powder to isopropanol is 1 g:22 mL - 28 mL.

[0018] As a preferred embodiment, the power of the ultrasonic wave is 700 - 750 W, and the ultrasonic extraction time is 60 min - 100 min.

[0019] As a preferred embodiment, a 0.22 μm organic filter membrane is used during filtration.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] For the present invention, isopropanol is added to the dry powder of vegetable plant tissue, and it is inverted up and down until the solid is completely dissolved. Then it is vortexed and placed in a 4°C environment for static placement in the dark. Subsequently, it is extracted at room temperature in an ultrasonic cleaner for 80 min, centrifuged, and the supernatant is aspirated and dried with a nitrogen blower. It is redissolved in n-hexane, allowed to stand, and filtered through a 0.22 μm organic filter membrane before being ready for machine detection. A high-performance liquid chromatograph is used, with a normal-phase chromatographic column (Hypersil GOLD Silica (250×4.6 mm, 5 μm)), a mobile phase of n-hexane: tert-butyl methyl ether = 94:6, a column temperature of 25°C, a flow rate of 0.6 ml / min, an injection volume of 6 μl, and an ultraviolet detection wavelength of 295 nm. The method for detecting vitamin E in vegetables provided by the present invention uses isopropanol as the extraction solvent, avoiding the disadvantages of easy volatilization and toxicity of using n-hexane. The required sample amount for detection is relatively small, only 0.2 g of the dried sample powder. The present invention can complete the detection of 8 VE isomers using a high-performance liquid chromatograph (normal phase), with relatively high detection efficiency and a short detection time, only about 20 min. Description of the Drawings

[0022] Figure 1 It is the high-performance liquid chromatography peak diagram of the mixed standard product of 8 VE isomers under different types of mobile phases of the present invention. Among them, Figure A is n-hexane, Figure B is n-hexane: tert-butyl methyl ether = 94:6 (volume ratio), and Figure C is isopropanol.

[0023] Figure 2 It is the high-performance liquid chromatography peak diagram of the mixed standard product of 8 VE isomers under different mobile phase ratios of the present invention. Among them, Figure A is the ratio of n-hexane: tert-butyl methyl ether of 90:10, Figure B is the ratio of n-hexane: tert-butyl methyl ether of 92:8, Figure C is the ratio of n-hexane: tert-butyl methyl ether of 94:6, Figure D is the ratio of n-hexane: tert-butyl methyl ether of 96:4, and Figure E is the ratio of n-hexane: tert-butyl methyl ether of 98:2.

[0024] Figure 3 It is the chromatogram of 8 VE isomers when different column temperatures are adopted in the present invention. Among them, Figure A is 15°C, Figure B is 20°C, Figure C is 30°C, and Figure D is 35°C.

[0025] Figure 4 It is the high-performance liquid chromatography peak diagram of the mixed standard product of 8 VE isomers when the mobile phase of the present invention is at different flow rates. Among them, Figure A is 0.4 mL / min, Figure B is 0.6 mL / min, Figure C is 0.8 mL / min, Figure D is 1.0 mL / min, and Figure E is 1.2 mL / min.

[0026] Figure 5This is the high-performance liquid chromatography peak diagram of an 8-VE isomers mixed standard under different sample injection volumes in the present invention. Among them, Diagram A is for 2 μl, Diagram B is for 6 μl, Diagram C is for 10 μl, Diagram D is for 14 μl, and Diagram E is for 18 μl.

[0027] Figure 6 This is the chromatogram of 8-VE isomers at different concentration gradients under the optimal chromatographic conditions of the present invention. Among them, Diagram A is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 2 μg / ml, Diagram B is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 4 μg / ml, Diagram C is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 8 μg / ml, Diagram D is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 16 μg / ml, Diagram E is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 32 μg / ml, Diagram F is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 64 μg / ml, and Diagram G is the HPLC diagram of the working solution of the VE isomers mixed standard with a concentration of 128 μg / ml.

[0028] Figure 7 This is the standard curve equation diagram of 8-VE isomers in the present invention. Among them, Diagram A is for α-Tocopherol, Diagram B is for α-3T Tocotrienol, Diagram C is for β-Tocopherol, Diagram D is for β-3T Tocotrienol, Diagram E is for γ-Tocopherol, Diagram F is for γ-3T Tocotrienol, Diagram G is for δ-Tocopherol, and Diagram H is for δ-3T Tocotrienol. Detailed implementation mode

[0029] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments, but the specific embodiments cited do not limit the present invention. The following test methods and detection methods are all conventional methods unless otherwise specified; the reagents and raw materials are all commercially available unless otherwise specified.

[0030] Aiming at the technical problems of the long detection time and narrow detection range caused by the use of normal-phase high-performance liquid chromatography in the prior art for the detection method of vitamin E, the present invention provides a detection method for vitamin E in vegetables, which can simultaneously detect 8-VE isomers in vegetables, with a short detection time and a wide detection range.

[0031] The technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] Example 1

[0033] A detection method for vitamin E in vegetables includes the following steps:

[0034] S1, Prepare a single standard working solution: Weigh the standards of α-Tocopherol, β-Tocopherol, γ-Tocopherol, δ-Tocopherol, α-3T Tocotrienol, β-3T Tocotrienol, γ-3T Tocotrienol, and δ-3T Tocotrienol respectively. Dissolve them in chromatographic pure n-hexane and make up the volume to prepare single standard stock solutions of α-Tocopherol, β-Tocopherol, γ-Tocopherol, δ-Tocopherol, α-3T Tocotrienol, β-3T Tocotrienol, γ-3T Tocotrienol, and δ-3T Tocotrienol with a concentration of 5 mg / ml each. Prepare single standard working solutions with a concentration of 128 μg / mL from the above 8 single standard stock solutions, and perform high-performance liquid chromatography analysis to record the retention times of the chromatographic peaks of the 8 vitamin E isomers respectively.

[0035] S2, Prepare a mixed standard working solution and make a standard curve: Take 256 μl of the single standard stock solutions of the 8 vitamin E isomers and mix them, add 7.952 mL of n-hexane, mix well to prepare a mixed standard working solution with a concentration of 128 μg / mL. Dilute it with n-hexane to obtain mixed standard solutions with concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL respectively. Perform high-performance liquid chromatography analysis on the mixed standard solutions, determine the corresponding relationship between the chromatographic peaks of the mixed standard solutions and the 8 vitamin E isomers according to the retention times of the chromatographic peaks, and record the peak areas of each vitamin E isomer. Draw standard curves for the 8 vitamin E isomers with the mass concentrations of the 8 vitamin E isomers as the abscissa and the peak areas as the ordinate.

[0036] S3, Treatment of the sample to be tested: Place the daylily flower buds in a forced-air drying oven at 60 °C and dry them to a constant weight. Grind them into powder with an ultra-high-throughput grinder, place them in a -40 °C environment and store them in the dark and sealed for later use. According to the dosage ratio of 1 g: 22 - 28 mL, add isopropanol to 0.2 g of the dry daylily flower bud powder, invert it up and down until the solid is completely dissolved, vortex for 30 s, then place it in a 4 °C environment and let it stand in the dark for 1 h, perform ultrasonic extraction at 700 - 750 W for 60 - 100 min, centrifuge to obtain the supernatant, aspirate the supernatant and dry it with a nitrogen blower, redissolve it in chromatographic pure n-hexane, let it stand, and filter it with a 0.22 μm organic filter membrane to obtain the sample solution to be tested.

[0037] S4, Sample Loading and Detection: Inject the sample solution to be measured into a special liquid chromatography injection vial, and use high performance liquid chromatography to detect the peak areas of 8 vitamin E isomers in the sample solution to be measured. Corresponding to the standard curves of the 8 vitamin E isomers, calculate the contents of the 8 vitamin E isomers; during detection, the liquid chromatography column is Hypersil GOLDSilica, with a specification of 250×4.6 mm, 5 μm; the mobile phase is a composition of n-hexane and tert-butyl methyl ether with a volume ratio of 94:6, the column temperature of the chromatography column is 25°C, the flow rate is 0.6 mL / min, the injection volume is 6 μL, and a UV detector with a detection wavelength of 295 nm is used to detect for 22 min.

[0038] Example 2

[0039] A method for detecting vitamin E in vegetables, comprising the following steps:

[0040] S1, The standard curves of 8 vitamin E isomers are the same as those in Example 1.

[0041] S2, Sample to be measured treatment: Place the daylily flower buds in a forced air drying oven at 60°C and dry to constant weight, grind them into powder with an ultra-high throughput grinder, place them in a -40°C environment for storage in the dark and sealed for later use. According to the dosage ratio of 1 g: 22 - 28 mL, add isopropanol to 0.2 g of dry daylily flower bud powder, invert up and down until the solid is completely dissolved, vortex for 30 s and then place in a 4°C environment for static storage in the dark for 1 h, carry out ultrasonic extraction at 700 - 750 W for 60 - 100 min, centrifuge to obtain the supernatant, aspirate the supernatant and dry it with a nitrogen blower, redissolve it in chromatographically pure n-hexane, let it stand, and filter it with a 0.22 μm organic filter membrane to obtain the sample solution to be measured.

[0042] S3, Sample Loading and Detection: Inject the sample solution to be measured into a special liquid chromatography injection vial, and use high performance liquid chromatography to detect the peak areas of 8 vitamin E isomers in the sample solution to be measured. Corresponding to the standard curves of the 8 vitamin E isomers, calculate the contents of the 8 vitamin E isomers; during detection, the liquid chromatography column is Hypersil GOLDSilica, with a specification of 250×4.6 mm, 5 μm; the mobile phase is a composition of n-hexane and tert-butyl methyl ether with a volume ratio of 94:6, the column temperature of the chromatography column is 20°C, the flow rate is 0.4 mL / min, the injection volume is 10 μL, and a UV detector with a detection wavelength of 295 nm is used to detect for 25 min.

[0043] Example 3

[0044] A method for detecting vitamin E in vegetables, comprising the following steps:

[0045] S1, The standard curves of 8 vitamin E isomers are the same as those in Example 1.

[0046] S2, Pretreatment of the sample to be tested: Place the daylily flower buds in a forced-air drying oven at 60 °C and dry them to a constant weight. Grind them into powder using an ultra-high-throughput grinder, store them in a light-proof and sealed container at -40 °C for later use. According to the dosage ratio of 1 g: 22 - 28 mL, add isopropanol to 0.2 g of dry daylily flower bud powder, invert it up and down until the solid is completely dissolved. After vortexing for 30 s, place it in a light-proof environment at 4 °C and let it stand for 1 h. Perform ultrasonic extraction at 700 - 750 W for 60 - 100 min, centrifuge to obtain the supernatant. Aspirate the supernatant and dry it with a nitrogen blower, then redissolve it in chromatographically pure n-hexane, let it stand, and filter it through a 0.22 μm organic filter membrane to obtain the sample solution to be tested.

[0047] S3, Sample injection and detection: Inject the sample solution to be tested into a special injection vial for liquid chromatography, and use high-performance liquid chromatography to detect the peak areas of 8 vitamin E isomers in the sample solution to be tested. Corresponding to the standard curves of the 8 vitamin E isomers, calculate the contents of the 8 vitamin E isomers; during detection, the liquid chromatography column is Hypersil GOLDSilica, with a specification of 250×4.6 mm, 5 μm; the mobile phase is a composition of n-hexane and tert-butyl methyl ether with a volume ratio of 94:6, the column temperature of the chromatographic column is 30 °C, the flow rate is 0.8 mL / min, the injection volume is 8 μL, and a UV detector with a detection wavelength of 295 nm is used for detection for 20 min.

[0048] The following specifically describes the detection process of the present invention.

[0049] 1 Sample pretreatment

[0050] Place the daylily flower buds in a forced-air drying oven at 60 °C and dry them to a constant weight. Grind them into powder using an ultra-high-throughput grinder, store them in a light-proof and sealed container at -40 °C for later use.

[0051] 2 Optimization of the vitamin E extraction system from daylily

[0052] (1) Required instruments and reagents

[0053] High-performance liquid chromatograph, model μltimate 3000, purchased from Thermo Fisher Company, USA; chromatographic column, model Hypersil GOLD Silica (250×4.6 mm, 5 μm); ultra-high-throughput grinder, model HODERn9980, purchased from Beijing Hede Company; digital display forced-air drying oven, model DHG-9240S, purchased from Ningbo Ledian Instrument Manufacturing Co., Ltd.; ultrasonic cleaner, model SB25-12DTD, purchased from Ningbo Xinzhi Biotechnology Co., Ltd., with a maximum power of 720 W; nitrogen blower, model HSC-12B, purchased from Tianjin Hengao Technology Development Co., Ltd.

[0054] n-Hexane (chromatographic grade), isopropanol (chromatographic grade), tert-butyl methyl ether (chromatographic grade), DMP (2,2-dimethoxypropane), tetrahydrofuran (chromatographic grade), anhydrous ethanol (analytical grade), acetone (analytical grade), n-hexane (analytical grade), isopropanol (analytical grade), ascorbic acid; α-T, β-T, γ-T, δ-T tocopherol standards, α-3T, β-3T, γ-3T, δ-3T tocotrienol standards, HPLC ≥ 98%, all purchased from Solebao Company.

[0055] (2) Extraction process overview: Accurately weigh 0.2 g of daylily flower bud powder, add ascorbic acid and extractant in proportion, turn it upside down until the solid is completely dissolved, vortex it on a vortex oscillator for 30 seconds, place it at 4°C and stand in the dark for 1 hour; extract it at room temperature in an ultrasonic cleaner, then use a centrifuge to centrifuge at 12000 r / min for 15 minutes, and absorb the supernatant; blow the supernatant dry with a nitrogen blower, dissolve it in 2 ml of n-hexane (chromatographic grade), stand for 1 hour, pass it through a 0.22 μm organic filter membrane, and put it on the machine for testing.

[0056] (3) Single factor optimization of extraction system

[0057] Optimized conditions: Extraction agent types: n-hexane, isopropanol, anhydrous ethanol, acetone. Liquid-to-solid ratio: 5:1, 15:1, 25:1, 35:1, 45:1. Ultrasonic time: 0min, 20min, 40min, 60min, 80min. Ultrasonic power: 288W, 396W, 504W, 612W, 720W. The α-T content of daylily buds was determined using the determined chromatographic conditions, and the α-tocopherol (α-T) content was used as an indicator to evaluate the different levels of each factor.

[0058] (4) Response surface optimization of extraction system

[0059] This experiment used the statistical analysis software Design-Expert 13.0. According to the Box-Behnken design principle (BB), the results of the single-factor experiment were comprehensively screened out to select three factors and three levels, and the response surface experiment was designed. The three main factors: liquid-to-solid ratio (A), ultrasonic time (B), and ultrasonic power (C) were used as independent variables, and the α-T content was used as the response value Y to obtain the best extraction parameters. The α-T content of daylily buds was determined using the optimized optimal chromatographic detection system, with three biological replicates. The α-tocopherol (α-T) content was used as an indicator to evaluate the different levels of each factor.

[0060] (5) Orthogonal experimental optimization of the extraction system

[0061] Using the statistical analysis software DPS, an orthogonal experimental design was carried out with the best factors screened by single-factor design. Three levels were set for each factor, and a suitable orthogonal experimental design table was selected. The optimized optimal chromatographic detection system was used to determine the α-T content in the daylily flower buds, with 3 biological replicates. The content of α-tocopherol (α-T) was used as an index to evaluate different levels of each factor.

[0062] (6) Comparison of the optimal extraction systems obtained by the response surface method and the orthogonal method

[0063] Using the optimal extraction conditions screened by the response surface test and the orthogonal experimental design, the difference in the α-T content under the optimal solution of the extraction system obtained from these two multi-factor design experiments was compared to determine the final VE extraction system for daylily flower buds.

[0064] (7) Determination and calculation of the content of vitamin E (α-tocopherol)

[0065] According to the standard curve equation of α-tocopherol, the concentration c of α-tocopherol in the test solution was calculated, and then the content A of α-tocopherol was obtained.

[0066] Calculation formula: A = c * v / m

[0067] Note: A is the content of α-tocopherol in the daylily flower bud sample (μg / g); c is the concentration of each tocopherol in the daylily flower bud sample (μg / ml); v is the total volume (ml) when the daylily flower bud sample is redissolved; m is the mass (g) of the daylily flower bud sample used for extraction.

[0068] (8) The finally determined optimal extraction system

[0069] Accurately weigh 0.2 g of dry daylily flower bud powder, add isopropanol according to the liquid-solid ratio of 25 ml / g, invert up and down until the solid is completely dissolved, vortex for 30 s, then place it in the dark at 4 °C for 1 h, then extract at room temperature for 80 min in an ultrasonic cleaner with 720 W, then use a centrifuge to centrifuge at 12000 r / min for 15 min, absorb the supernatant and dry the supernatant with a nitrogen blower, redissolve it in 2 ml of n-hexane, let it stand for 1 h, and then filter it through a 0.22 μm organic filter membrane, and then it can be used for on-machine detection.

[0070] (9) Evaluation of the optimal extraction system

[0071] Repeatability evaluation: Select the same batch of dry daylily flower bud powder, divide it evenly into 5 parts, and extract simultaneously according to the optimized extraction method, and perform 3 repeated determinations under the determined chromatographic conditions. The results are shown in Table 1. The RSD of each VE isomer is 0.929, indicating that this extraction method has good reproducibility and can be used for the quantitative analysis of each VE isomer.

[0072] Table 1 Results of repeated extraction

[0073]

[0074] Stability evaluation: Select the dried powder of the same batch of daylily flower buds, evenly divide it into 5 parts, extract simultaneously according to the optimized extraction method, redissolve the extracted dry samples at 0h, 2h, 4h, 8h, 14h, and 24h, load them onto the machine, and measure. The results are shown in Table 2. The RSD of each isomer ≤ 0.946, indicating that the samples under this extraction condition are relatively stable and can be used for the detection of VE isomers.

[0075] Table 2 Results of stability determination

[0076]

[0077] 3 Optimization of the vitamin E detection system for daylily

[0078] (1) Instruments and reagents required

[0079] High-performance liquid chromatograph, model Ultimate 3000, purchased from Thermo Fisher Company, USA; chromatographic column, model Hypersil GOLD Silica (250×4.6 mm, 5 μm); n-hexane (chromatographically pure), isopropanol (chromatographically pure), tert-butyl methyl ether (chromatographically pure); α-T, β-T, γ-T, δ-T tocopherol standards, α-3T, β-3T, γ-3T, δ-3T tocotrienol standards, HPLC ≥ 98%, all purchased from Solarbio Company. All standards were prepared as single standard stock solutions with a concentration of 5 mg / ml.

[0080] (2) The optimized conditions include:

[0081] Types of mobile phases ( Figure 1 ): n-hexane, n-hexane:tert-butyl methyl ether = 94:6 (volume ratio), isopropanol. Proportions of mobile phases ( Figure 2 ): The proportion of n-hexane:tert-butyl methyl ether is 90:10, 92:8, 94:6, 96:4, 98:2. Column temperature ( Figure 3 ): 15°C, 20°C, 25°C, 30°C, 35°C. Flow rate ( Figure 4 ): 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, 1.0 mL / min, 1.2 mL / min. Injection volume ( Figure 5 ): 2 μl, 6 μl, 10 μl, 14 μl, 18 μl.

[0082] The finally determined optimal detection conditions are as follows: mobile phase, n-hexane: tert-butyl methyl ether = 94:6; column temperature, 25°C; flow rate, 0.6 ml / min; injection volume, 6 μl; UV detection wavelength, 295 nm; overall analysis time, approximately 25 min.

[0083] (3) Establishment of the calibration curve under the optimal detection system

[0084] Prepare 7 concentration gradient standard mixed solutions of each VE isomer, and analyze them under the selected optimal chromatographic conditions. Using the concentrations (X, μg / ml) of 8 VE isomers as the abscissa and the peak areas (Y) as the ordinate, perform regression fitting using Excel software to obtain the standard curve equations of each VE isomer. The results are shown in Table 3 below. Figure 6 and Figure 7 . Under these chromatographic conditions, the R 2 of the linear equations of 8 VE isomers is ≥ 0.9997, indicating a good linear relationship.

[0085] Table 3 Relationship between the concentration X and peak area Y of each VE isomer

[0086] Tocopherol Regression equation <![CDATA[R 2 > α-T y = 0.0863x - 0.0201 0.9999 α-3T y = 0.0763x - 0.0397 1 β-T y = 0.0771x + 0.0087 0.9997 β-3T y = 0.0796x - 0.0491 0.9999 γ-T y = 0.0882x + 0.0339 0.9999 γ-3T y = 0.1362x - 0.0606 1 δ-T y = 0.0612x - 0.0141 1 δ-3T y = 0.071x - 0.0199 1

[0087] (4) Evaluation of the optimal detection system

[0088] System suitability evaluation: Perform detection under the optimized chromatographic conditions, inject samples repeatedly 3 times, and count and calculate the theoretical plate number N. The results show that the theoretical plate number of each isomer that can be completely separated is ≥ 16274, indicating strong column efficiency and good system suitability, as shown in Table 4.

[0089] Table 4 Theoretical plate numbers of 8 VE isomers under the optimal detection system

[0090]

[0091]

[0092] Precision evaluation: Take the standard mixture solution of the same concentration, inject samples repeatedly 6 times, record the peak areas of each VE isomer each time, and calculate the relative standard deviation (RSD) of the peak areas of each isomer. The results show that the RSD of the mixed standard solution of each VE isomer for 6 injections is ≤ 0.60, as shown in Table 5, indicating high precision of this chromatographic condition and instrument, which can be used for the quantitative analysis of each VE isomer.

[0093] Table 5 Results of precision determination

[0094]

[0095]

[0096] Stability evaluation: Take the standard product mixed solution with the same concentration and inject samples for determination at 0h, 2h, 4h, 6h, and 8h respectively. Record the peak areas of each VE isomer each time, and calculate the RSD values of the peak areas of each isomer. The results show that the RSD of each VE isomer ≤ 1.82, as shown in Table 6, indicating that HPLC under these chromatographic conditions is relatively stable and can be used for the detection of VE isomers.

[0097] Table 6 Results of stability determination

[0098]

[0099]

[0100] Summary of the optimal methods for extracting and detecting vitamin E from daylily

[0101] Place the daylily flower buds in a forced-air drying oven at 60 °C and dry them to a constant weight. Grind them into powder using an ultra-high throughput grinder, place them in a -40 °C environment, store them in the dark and sealed for later use. Accurately weigh 0.2 g of the dry powder of daylily flower buds, add isopropanol according to the liquid-solid ratio of 25 mL / g, invert up and down until the solid is completely dissolved. After vortex oscillation for 30 s, place it in a 4 °C environment, let it stand in the dark for 1 h, then extract it at room temperature for 80 min in an ultrasonic cleaner with 720 W. Then use a centrifuge to centrifuge at 12000 r / min for 15 min, aspirate the supernatant, and dry the supernatant with a nitrogen blower. Re-dissolve it in 2 mL of n-hexane, let it stand for 1 h, and then filter it through a 0.22 μm organic filter membrane, and then it can be injected into the instrument for detection. Use a high performance liquid chromatograph, a normal-phase chromatographic column (Hypersil GOLD Silica (250×4.6 mm, 5 μm)), the mobile phase is n-hexane: tert-butyl methyl ether = 94:6, the column temperature is 25 °C, the flow rate is 0.6 mL / min, the injection volume is 6 μL, and the ultraviolet detection wavelength is 295 nm.

[0102] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and deformations.

Claims

1. A method for detecting vitamin E in vegetables, characterized in that, It includes the following steps: Sample to be tested treatment: Add isopropanol to the dry powder of vegetable plant tissue, dissolve it, extract by ultrasonic wave, obtain the supernatant, dry it, redissolve it in n-hexane, filter it, and obtain the sample solution to be tested; Sample injection and detection: Use high performance liquid chromatography to detect the peak areas of 8 vitamin E isomers in the sample solution to be tested, correspondingly combine with the standard curves of the 8 vitamin E isomers, and calculate the contents of the 8 vitamin E isomers; When detecting, the liquid chromatography column is Hypersil GOLD Silica, with the specification of 250×4.6mm, 5μm; The mobile phase is a composition of n-hexane and tert-butyl methyl ether with a volume ratio of 94:6, the column temperature of the chromatographic column is 20°C to 30°C, the flow rate is 0.4mL / min to 0.8mL / min, and the sample injection volume is 6μL to 10μL.

2. The detection method of vitamin E in vegetables according to claim 1, wherein The detection temperature of the liquid chromatography column is 25°C.

3. The detection method of vitamin E in vegetables according to claim 1, characterized in that, The flow rate of the mobile phase is 0.6mL / min.

4. The detection method of vitamin E in vegetables according to claim 1, characterized in that, The sample injection volume is 6μL.

5. The detection method of vitamin E in vegetables according to claim 1, wherein The liquid chromatography adopts an ultraviolet detector for detection, and the detection wavelength of the ultraviolet detector is 295nm.

6. The detection method of vitamin E in vegetables according to claim 1, wherein, The detection time is 20min to 25min.

7. The detection method of vitamin E in vegetables according to claim 1, characterized in that The dosage ratio of the dry powder of vegetable plant tissue to isopropanol is 1g: 22mL to 28mL.

8. The detection method of vitamin E in vegetables according to claim 1, characterized in that, The power of the ultrasonic wave is 700W to 750W, and the ultrasonic extraction time is 60min to 100min.

9. The detection method of vitamin E in vegetables according to claim 1, characterized in that, When filtering, a 0.22μm organic filter membrane is used.

10. The detection method of vitamin E in vegetables according to claim 1, characterized in that, The method for preparing the standard curves of the 8 vitamin E isomers includes the following steps: Respectively prepare single standard working solutions of 8 vitamin E isomers, analyze them by high performance liquid chromatography, and respectively record the chromatographic peak retention times of the 8 vitamin E isomers; Respectively take the single standard stock solutions of 8 vitamin E isomers, mix them, add n-hexane, prepare a mixed standard working solution, dilute it, and respectively obtain mixed standard solutions with concentrations of 2μg / mL to 64μg / mL. Analyze the mixed standard solutions by high performance liquid chromatography, and determine the corresponding relationship between the chromatographic peaks of the mixed standard solutions and the 8 vitamin E isomers according to the retention times of the chromatographic peaks, and respectively draw the standard curves of the 8 vitamin E isomers.

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