A method for detecting the separation of diglycerides in vegetable oils and their qualitative analysis

Abstract

The application discloses a detection method for separating diglyceride in vegetable oil and qualitative analysis of the diglyceride, and the diglyceride in the vegetable oil is dissolved by using n-hexane to obtain a sample for standby; different proportion eluent is used to wash and elute a solid phase extraction column, and eluent is collected and dried for standby to obtain eluate; the eluate is analyzed and quantified by using a thin layer chromatography and gas chromatography, and finally, the eluate is finely qualitatively analyzed by using a triple quadrupole; the separation method is simple, the separation of the diglyceride in the vegetable oil can be realized by using two extraction cartridges, the test steps are simplified, the separation purity is high, the cost and time are reduced; finally, qualitative analysis is performed by using GC-MS, the analysis time is short, the specific fatty acid composition in the diglyceride is more finely analyzed, and the qualitative analysis detection of food nutrition and the guidance of related food production are facilitated.

Description

Technical Field

[0001] This invention relates to the field of detection, and more particularly to a method for separating diglycerides from vegetable oils and their qualitative analysis. Background Technology

[0002] Edible oils are one of the main raw materials in the food industry and one of the three major energy-producing substances required by the human body. Structured lipids, also known as structural lipids, are triglycerides with different functions by altering the composition and positional distribution of fatty acids directly linked to the glycerol backbone. They possess unique metabolic pathways, nutritional value, and physicochemical properties, maximizing the nutritional and functional properties of oils and fats, and have enormous application potential in the food, pharmaceutical, and other fields.

[0003] Structured lipids were initially synthesized chemically, primarily through esterification or transesterification reactions to obtain the target products, followed by purification using physical methods (such as crystallization and distillation) to achieve high purity. In recent years, with the development of bioengineering technology, enzymatic synthesis of structured lipids has attracted increasing attention. Unlike traditional physical and chemical methods, enzymatic reactions offer milder and more selective conditions, providing a more efficient means of synthesizing structured lipids. Enzymatic synthesis of structured lipids includes esterification, transesterification, acid hydrolysis, and two-step methods. Esterification is mainly used for synthesizing monoesters and diesters, while the synthesis of triglycerides requires separation and purification methods such as molecular distillation and crystallization, making the process relatively complex.

[0004] The enzymatic hydrolysis products of triglycerides mainly include diglycerides (DAG), monoglycerides (MAG), fatty acids (FFA), and glycerol. According to domestic and international literature, thin-layer chromatography (TLC) is currently the primary method for separating the monoglyceride components from triglyceride hydrolysis products. TLC requires the preparation of thin-layer plates using silica gel G, the separation of hydrolysis products using a specific ratio of developing solvent, followed by color development of the thin-layer plate, scraping off the silica gel, and extraction of monoglycerides with an organic solvent. This process is relatively complex, and the skill level of the operator significantly affects the separation and extraction results. Solid-phase extraction (SPE), on the other hand, offers advantages such as low organic solvent consumption, high enrichment factor, minimal environmental pollution, effective sample purification and pre-separation of interfering components, easy sample preservation, simple operation, convenient batch sample processing, and high speed. Therefore, this invention experimentally studies the separation of components from triglyceride hydrolysis products using a Florisil SPE column, verifying the practicality of using a Florisil SPE column for separating triglycerides and diglycerides.

[0005] For example, Chinese invention patent application number CN202011096697.9 discloses a rapid detection method for glycidol and glycidyl diester in edible vegetable oil, which includes centrifuging the sample, acidification reaction, centrifugation again and derivatization reaction, and finally putting it into GC-MS for detection and analysis, and quantification. Although both this invention and the previous one involve detecting components in vegetable oils, the specific components, glycidyl diglyceride and diglyceride, have completely different structures and functions. The glycidyl diglyceride in the previous application is genotoxic and can induce malignant tumors after use, thus posing a potential safety hazard. The International Agency for Research on Cancer (IARC) classifies it as a Group 2A carcinogen, and the German Society for Oil Science classifies it as a Group 2 carcinogen. Glycidyl diglyceride is present in relatively small amounts in vegetable oils and exhibits certain thermal instability. In contrast, the diglyceride in this application possesses unique metabolic pathways, nutritional value, and physicochemical properties, maximizing the nutritional and functional benefits of oils. It has significant potential applications in food, medicine, and other fields, specifically providing energy, aiding in the absorption of vitamins and amino acids, and enhancing nutrient absorption capacity; therefore, it is a beneficial substance for the human body. Thus, the research subjects are two substances with completely different physicochemical properties. Furthermore, the previous application focuses more on the quantitative analysis of glycidyl diglyceride, while this application focuses more on the qualitative analysis of diglyceride, and the separation processes are also entirely different.

[0006] Currently, there are very few methods available on the market that offer effective and rapid separation of diglycerides, as well as methods for rapid and accurate qualitative analysis of diglycerides using triple quadrupole.

[0007] In view of this, it is indeed necessary to provide a technical solution to the above-mentioned technical problems. Summary of the Invention

[0008] This invention provides a method for separating diglycerides from vegetable oils and performing qualitative analysis thereon. The separation is effective and convenient, and the triple quadrupole method enables rapid and accurate qualitative analysis of diglycerides.

[0009] The present invention solves its technical problem by adopting the following technical solution:

[0010] A method for separating diglycerides from vegetable oils and their qualitative analysis, characterized by comprising the following steps:

[0011] S1: Dissolve the vegetable oil in n-hexane to obtain a sample for later use; equilibrate the packing material with n-hexane to obtain a solid-phase extraction column;

[0012] S2: Control the flow rate of the sample from step S1 and add it to the solid phase extraction column; wash the solid phase extraction column with eluent of different ratios, collect the eluent and dry it for later use.

[0013] S3: The eluent from step S2 is reconstituted in n-hexane, mixed well, and then the eluted sample is spotted and placed in a chromatography tank for thin-layer chromatography to obtain the colorimetric results; the eluent is derivatized by hydroxysilanization to obtain GC analysis and GC chromatogram.

[0014] S4: The eluent separated in step S3 is qualitatively analyzed by GC-MS. The eluent is separated on a capillary column with helium as the carrier gas at a flow rate of 1-3 mL / min. The initial temperature is set at 150-300℃ and held for 1-4 min. The temperature is then increased to 250-450℃ at a rate of 5-20℃ / min and held for 2-5 min. The temperature is then increased to 300-350℃ at a rate of 5-15℃ / min and held for 15-20 min. The injector temperature is 250-400℃, the split ratio is (15-30):(1-2), and the injection volume is 0.5-3 μL. The MS settings include using 70 eV electron impact ionization as the ion source, collecting and analyzing ions in the range of 200-650 m / z, and using an emission current of 60-80 μA to analyze the properties of the eluent components.

[0015] As a preferred embodiment, in step S1, the mass of vegetable oil is weighed to be 20-100 mg, and the mass concentration of the sample dissolved in n-hexane is controlled to be 5 mg / mL-50 mg / mL.

[0016] As a preferred embodiment, in step S1, the packing material includes one of alumina, silica gel, and Florisil; the volume of n-hexane is 3-10 times that of the packing material for column equilibration.

[0017] As a preferred embodiment, in step S2, the flow rate is 1-5 mL / min, and the sample volume is 0.5-1 times the volume of the packing material in the solid-phase extraction column.

[0018] As a preferred embodiment, in step S2, the elution separation includes eluting with 100% hexane to obtain triglycerides; and eluting with a hexane to ethyl acetate ratio of (25-85):(15-75) to obtain diglycerides.

[0019] As a preferred embodiment, in step S2, the eluent includes diglyceride eluent and triglyceride eluent, wherein the diglyceride eluent is obtained by elution through two columns.

[0020] As a preferred embodiment, in step S3, the mass concentration of the eluent redissolved in n-hexane is controlled at 10 mg / mL-30 mg / mL; the developing solvent is n-hexane, anhydrous diethyl ether and glacial acetic acid in a ratio of (40-50):(50-55):(1-2).

[0021] As a preferred embodiment, in step S3, the GC analysis instrument includes an Agilent 6890 gas chromatograph with an FID detector; the chromatographic column is a DB-5HT, 15m × 0.25mm × 0.1μm; the injection volume is 1μL; the split ratio is 20:1; the initial programmed temperature is 120℃, held for 1 min; the temperature is increased to 300℃ at a rate of 20℃ / min and held for 3 min; the temperature is increased to 380℃ at a rate of 10℃ / min and held for 10 min to obtain the GC chromatogram.

[0022] As a preferred embodiment, in step S3, the derivatization process is as follows:

[0023] S11: Dissolve the eluent from step S2 in pyridine, shake for 1-2 seconds to obtain a mixture; the derivatizing reagents include BSTFA and TMCS;

[0024] S12: Add the derivatizing reagent to the mixture and mix well. Place the mixture in a constant temperature oven at 70℃ for 30 minutes to complete the derivatization reaction.

[0025] As a preferred embodiment, in step S4, the eluent separated in step S3 is qualitatively analyzed by GC-MS. The eluent is separated on a capillary column with helium as the carrier gas and a flow rate of 1 mL / min. The initial temperature is set to 200℃ and the holding time is 1 min. The temperature is then increased to 300℃ at a rate of 20℃ / min and held for 3 min. The temperature is then increased to 320℃ at a rate of 10℃ / min and held for 19 min. The injector temperature is 300℃, the split ratio is 20:1, and the injection volume is 1 μL. The MS settings include using 70 eV electron impact ionization as the ion source, collecting and analyzing ions in the range of 200-650 m / z, and an emission current of 80 μA to analyze the properties of the eluent components.

[0026] The vegetable oil includes one of flaxseed oil, peanut oil, and corn oil.

[0027] Preferably, the vegetable oil includes flaxseed oil; the filler includes Florisil.

[0028] The GC-MS results were analyzed qualitatively and quantitatively using Agilent MassHunter software.

[0029] The beneficial effects of this invention are as follows: This invention provides a method for separating diglycerides from vegetable oils and their qualitative analysis. Through simple steps such as sample preparation, column activation and loading, and elution, diglycerides in vegetable oils can be separated using two extraction columns. This simplifies the experimental steps, achieves high separation purity, and reduces cost and time. Finally, through optimized qualitative detection using GC-MS, targeted and efficient analysis of the diglyceride components in vegetable oils is performed. The analysis time is stabilized at 20-30 minutes, while providing a more precise analysis of the specific fatty acid composition of the diglycerides. This is beneficial for the qualitative analysis and detection of food nutrition and for guiding related food production. Attached Figure Description

[0030] Figure 1 Chromatogram of total ion esters in flaxseed oil;

[0031] Figure 2 TLC results of flaxseed oil eluent;

[0032] Figure 3 Figure 1 shows the GC-FID results of two separate purification processes of flaxseed oil diglycerides. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0034] Example

[0035] A method for separating diglycerides from vegetable oils and their qualitative analysis includes the following steps:

[0036] S1: Prepared flaxseed oil with a diglyceride to triglyceride ratio of 6:4 was obtained, dissolved in n-hexane, and the mass concentration was controlled at 15 mg / mL. The sample was then stored in a -20℃ refrigerator for later use. A solid-phase extraction column was used with 8 times the packing volume of n-hexane and Florisil as the packing material.

[0037] S2: Add a sample volume equal to 1 packing volume from step S1 to a solid-phase extraction column at a flow rate of 3 mL / min. Elute the solid-phase extraction column with 100% hexane to elute the triglycerides adsorbed on the column. Elute the solid-phase extraction column with a hexane:ethyl acetate mixture of different polarities (70:30) to elute the diglycerides adsorbed on the column. Dry the collected diglyceride eluent under nitrogen evaporation and add it to the activated second solid-phase extraction column. Repeat the above procedure once more, collect the diglyceride eluent, and dry it under nitrogen for later use.

[0038] S3: Redissolve the eluent from step S2 in n-hexane, maintaining a concentration of 15 mg / mL, to obtain the eluted sample. Spot the eluted sample onto a silica gel plate and place it in a chromatography tank for thin-layer chromatography. The developing solvent used is n-hexane:anhydrous diethyl ether:glacial acetic acid in a volume ratio of 45:25:1. After air-drying, place the plate in iodine vapor for color development until the spots are fully visible. Observe whether triglycerides and diglycerides have been separated (see...). Figure 2 The eluent collected in step S2 was derivatized. The type of eluent was determined based on the TLC results. For the derivatization of fats, hydroxysilanization was used. The eluent from step S2 was dissolved in pyridine, mixed, and then BSTFA and TMCS derivatization reagents were added and mixed. The mixture was incubated at 70℃ for 30 min, filtered through an organic filter membrane, and finally analyzed by GC. The GC instrument was an Agilent 6890 gas chromatograph with an FID detector. The column was a DB-5HT, 15m × 0.25mm × 0.1μm. The injection volume was 1μL. The split ratio was 20:1. The temperature program was as follows: initial temperature 120℃, hold for 1 min; 120℃~300℃, heating rate 20℃ / min, hold for 3 min; 300℃~380℃, heating rate 10℃ / min, hold for 10 min. The GC chromatogram was obtained, the data were analyzed, and the percentage content of each component was calculated (see...). Figure 3 );

[0039] S4: The eluent separated in step S3 was qualitatively analyzed by GC-MS. The eluent was separated on a capillary column with helium as the carrier gas at a flow rate of 1 mL / min. The initial temperature was set at 200℃ and held for 1 min; the temperature was then increased to 300℃ at a rate of 20℃ / min and held for 3 min; then increased to 320℃ at a rate of 10℃ / min and held for 19 min. The injector temperature was set to 300℃, the split ratio to 20:1, and the injection volume to 1 μL. The MS settings included using a 70 eV electron shock ionization source and collecting and analyzing ions from 200 to 650 eV. m / z Ions within the range, with an emission current of 80 μA, were analyzed to obtain the properties of the eluent components (see...). Figure 1 ).

[0040] This invention utilizes homemade flaxseed oil with a diglyceride to triglyceride ratio of 1:6:3, and... Figure 2 , Figure 3 The comparison shows that the purity of the separated diglycerides can reach over 90%, therefore the method for separating diglycerides from vegetable oils in this invention is highly efficient and useful; the diglyceride products obtained by the above separation are further analyzed by triple quadrupole analysis, as shown in Table 1.

[0041] Table 1 Qualitative Analysis of Diglycerides

[0042]

[0043] The chromatographic peak with a retention time of 10-16 min in the total ion chromatogram represents the target diglyceride, according to [M-RCO2]. + [RCO2+58] + [M-RCO2CH2] + [RCO] + [M-89] + [M-15] + These characteristic ion fragments can be used to infer the fatty acid composition of diglycerides.

[0044] As shown in the table, 1-linoleic acid-3-linolenic acid diglyceride has the highest content among flaxseed oil diglycerides, followed by 1,3-dialinonic acid diglyceride. The lowest content is 1,3-dispalmitoyl diglyceride, followed by 1-palmitoyl-2-linolenic acid diglyceride, which is consistent with the results of flaxseed oil fatty acid content analysis. Overall, in the flaxseed oil diglyceride isomers, 1,2-diglyceride accounts for 15.55%, and 1,3-diglyceride accounts for 84.45%, with a ratio of approximately 1:5.43.

[0045] Therefore, this invention provides a more precise qualitative analysis of diglycerides in vegetable oils, revealing the specific fatty acid composition, accurate related isomers, and component proportions. The analysis time is consistently between 20-30 minutes, which is short and efficient, thus facilitating qualitative analysis and detection of food nutrition and guiding related food production.

[0046] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for separating diglycerides from vegetable oils and their qualitative analysis, characterized in that, Includes the following steps: S1: Dissolve the vegetable oil in n-hexane to obtain a sample for later use; equilibrate the Florisil packing material with n-hexane to obtain a Florisil solid-phase extraction column. S2: Control the flow rate of the sample from step S1 and add it to a Florisil solid-phase extraction column; first, elute with 100% n-hexane to obtain triglyceride eluent, then elute with n-hexane and ethyl acetate in a ratio of (70-85):(15-30) to obtain diglyceride eluent; add the diglyceride eluent to a second Florisil solid-phase extraction column and repeat the same elution process, collect the diglyceride eluent, dry it for later use, and obtain the eluent; S3: The eluent from step S2 is redissolved in n-hexane to obtain an eluted sample. The eluted sample is spotted and placed in a chromatography tank for thin-layer chromatography to obtain a colorimetric result. The eluent from step S2 is derivatized by hydroxysilanization and analyzed by GC to obtain a GC chromatogram. S4: The diglyceride eluent separated in step S2 was qualitatively analyzed by GC-MS. The diglyceride eluent was separated on a capillary column with helium as the carrier gas at a flow rate of 1 mL / min. The initial temperature was set to 200℃ and held for 1 min; the temperature was then increased to 300℃ at a rate of 20℃ / min and held for 3 min; then increased to 320℃ at a rate of 10℃ / min and held for 19 min. The injector temperature was 300℃, the split ratio was 20:1, and the injection volume was 1 μL. The MS settings included using 70 eV electron shock ionization as the ion source and collecting and analyzing 200-650 μL of ions. m / z Ions within the range, with an emission current of 80 μA, were analyzed to determine the properties of the diglyceride eluent components; The derivatization process is as follows: S11: Dissolve the eluent from step S2 in pyridine, shake for 1-2 seconds to obtain a mixture; the derivatizing reagents include BSTFA and TMCS; S12: Add the derivatizing reagent to the mixture and mix well. Place the mixture in a constant temperature oven at 70℃ for 30 minutes to complete the derivatization reaction.

2. The method for separating diglycerides from vegetable oils and their qualitative analysis according to claim 1, characterized in that, In step S1, 20-100 mg of vegetable oil is weighed, and the mass concentration of the sample dissolved in n-hexane is controlled between 5 mg / mL and 50 mg / mL.

3. The method for separating diglycerides from vegetable oils and their qualitative analysis according to claim 1, characterized in that, In step S1, the volume of n-hexane is 3-10 times that of the Florisil packing for column equilibration.

4. The method for separating diglycerides from vegetable oils and their qualitative analysis according to claim 1, characterized in that, In step S3, the mass concentration of the eluent redissolved in n-hexane is controlled between 10 mg / mL and 30 mg / mL.

5. The method for separating diglycerides from vegetable oils and their qualitative analysis according to claim 1, characterized in that, In step S3, the GC analysis instrument includes an Agilent 6890 gas chromatograph with an FID detector; the chromatographic column is a DB-5HT, 15 m × 0.25 mm × 0.1 μm; the injection volume is 1 μL; the split ratio is 20:1; the initial programmed temperature is 120℃, held for 1 min; the temperature is increased to 300℃ at a rate of 20℃ / min and held for 3 min; the temperature is increased to 380℃ at a rate of 10℃ / min and held for 10 min to obtain the GC chromatogram.

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