A method for detecting multiple functional structured lipids in infant biscuits and application thereof

Abstract

The application provides a kind of detection method and application of multiple functional structural lipids in infant biscuit, belong to food detection technical field. Including: after sample is pulverized, using n-hexane-isopropyl alcohol-methanol-1,4-dioxane mixed extraction liquid extraction, obtain crude extract; Using modified graphene / silica gel composite adsorbent for solid phase extraction purification, obtain the test solution; Using high performance liquid chromatography-double detector combination system for detection, wherein C18 chromatographic column is combined with optimized gradient elution program to realize baseline separation of three C52 structural lipids, separation degree is greater than or equal to 1.5, ultraviolet detector and evaporation light scattering detector are connected in series respectively for determination of high concentration and low concentration target. The application realizes simultaneous detection of OPO, OPL and LPL three functional structural lipids in infant biscuit for the first time, has the advantages of comprehensive detection material, complex applicable matrix, excellent separation effect, high sensitivity, simple operation and the like.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting multiple functional structural lipids in infant biscuits and its application. Background Technology

[0002] Functional structural lipids are a class of lipid compounds that, through artificial design or modification, precisely regulate the fatty acid chains (such as carbon chain length, saturation, and double bond positions) and the connection positions of fatty acids on the glycerol backbone of triglyceride molecules. This allows them to possess physiological activities exceeding the basic nutritional functions (energy supply and storage) of ordinary lipids, and to act directionally on human metabolism and health regulation. Among them, 1,3-dioleoyl-2-palmitoylglycerol (OPO), 1-oleic-2-palmitoyl-3-linoleic acid triglyceride (OPL), and 1-linoleic-2-palmitoyl-3-linoleic acid triglyceride (LPL) possess characteristics that mimic the structure of breast milk fat. They are important nutrients in infant formula, promoting the absorption of fatty acids and minerals, reducing calcium soap formation, preventing constipation, and improving nutrient utilization. They are widely used in complementary food products such as infant biscuits. Therefore, establishing a precise and efficient method for determining various functional structural lipids in complementary foods such as infant biscuits is of great significance.

[0003] Currently, the determination of functional structured lipids in food mainly relies on the national standard GB 30604-2015, "National Food Safety Standard for Food Fortifiers: 1,3-Dioleoyl-2-palmitoylglycerol Triglycerides." This standard only controls the quality of pure OPO fortifiers, with the detection indicators being OPO content and the proportion of palmitic acid at the SN-2 position. It does not cover the detection of OPL and LPL, and is not applicable to final products such as infant biscuits. Furthermore, the gas chromatography method used in this standard requires saponification and methyl esterification, making it impossible to directly identify and distinguish intact triglyceride isomers, and even more difficult to achieve simultaneous detection of the three C52 structured lipids: OPO, OPL, and LPL.

[0004] Simultaneous detection of OPO and OPL has been attempted. For example, Chinese patent application CN121453956A discloses a method for detecting specific triglycerides in infant formula, using a CP-TAP CB capillary column combined with programmed temperature rise to achieve effective separation of OPO and OPL on a gas chromatography platform. However, this method still has the following shortcomings: First, the range of detectable substances is limited, covering only OPO and OPL, excluding other important C52-structured lipids such as LPL; second, the applicable matrix is ​​infant formula, and for complementary food products containing starch such as infant biscuits, the starch network structure encapsulates the fat, resulting in low extraction efficiency and easy loss of target substances when directly using this method for fat extraction; third, this method uses gas chromatography, which operates at high temperatures (up to 350℃), which may pose a risk of degradation for lipids with poor thermal stability; fourth, this method uses only a single FID detector, which has limited sensitivity for detecting low-content target substances.

[0005] Therefore, it is of great significance to develop a detection method that can simultaneously detect three C52-structured lipids, namely OPO, OPL, and LPL, which is suitable for complex starch-containing matrices such as infant biscuits, and has high detection sensitivity and is easy to operate. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for detecting multiple functional structural lipids in infant biscuits and its application. The method includes: pulverizing the sample and extracting it with a hexane-isopropanol-methanol-1,4-dioxane mixed extract to obtain a crude extract; purifying the crude extract using a modified graphene / silica gel composite adsorbent via solid-phase extraction (SPE) to obtain the analyte; and performing detection using a high-performance liquid chromatography-dual-detector system. The C18 column, combined with an optimized gradient elution program, achieves baseline separation of the three C52 structural lipids with a resolution ≥ 1.5. A UV detector and an evaporative light scattering detector are connected in series for the determination of high and low concentrations of the target analytes, respectively. This invention achieves, for the first time, the simultaneous detection of three functional structural lipids—OPO, OPL, and LPL—in infant biscuits, offering advantages such as comprehensive detection capabilities, applicability to complex matrices, excellent separation performance, high sensitivity, and ease of operation.

[0007] On one hand, the present invention provides a purification material for detecting multiple functional structural lipids in infant biscuits, including a modified graphene / silica composite adsorbent, wherein the modified graphene / silica composite adsorbent is obtained by reacting graphene oxide with tetramethoxysilane to form a primary product of graphene / silica composite adsorbent, and then modifying it with 3-aminopropyltrimethoxysilane.

[0008] The specific preparation method of the modified graphene / silica composite adsorbent is as follows: Weigh 0.4 g of graphene oxide material and add 100 mL of distilled water. Vortex for 4 h to form a suspension. Slowly add 200 mL of anhydrous ethanol solution and stir for 20 min. Slowly add ammonia solution to adjust the pH to 9.5. Slowly add 20 mL of tetramethoxysilane and stir at 50 °C for 12 h to form primary graphene / silica composite adsorbent particles. Filter, wash with anhydrous ethanol 3-5 times, and vacuum dry at 60 °C to constant weight to obtain the primary modified graphene / silica composite adsorbent. Take 5 g of the above material, add 50 mL of toluene and 1 mL of 3-aminopropyltrimethoxysilane, reflux at 105 °C for 8 h, cool and filter, and vacuum dry at 60 °C to constant weight to obtain the modified graphene / silica composite adsorbent. This material combines the high specific surface area of ​​graphene and the adsorption stability of silica. By introducing hydrophobic groups through modification, it can selectively adsorb impurities such as proteins, phospholipids, and starch in the sample, while avoiding the adsorption of target substances. Compared with traditional silica adsorbents, the impurity removal rate and target substance recovery rate are greatly improved.

[0009] On the other hand, the present invention provides a kit for detecting multiple functional structural lipids in infant biscuits, comprising the above-mentioned purification materials.

[0010] Furthermore, the kit also includes a mixed extraction solution composed of a mixture of hexane, isopropanol, methanol, and 1,4-dioxane, wherein the ratio of the hexane, isopropanol, methanol, and 1,4-dioxane mixture is 4~6:3~4:1:0.2.

[0011] Preferably, the volume ratio of n-hexane, isopropanol, methanol, and 1,4-dioxane is 5:3:1:0.2.

[0012] The four-component compound solvent system consisting of n-hexane, isopropanol, methanol, and 1,4-dioxane can efficiently extract various functional lipid structures with large polarity differences in infant biscuits. 1,4-dioxane, as a co-solvent, can improve the extraction effect of lipids encapsulated in starch network structure and inhibit the dissolution of impurities. Compared with traditional single extraction solutions, the extraction efficiency is increased by more than 10%.

[0013] Furthermore, this invention provides a method for simultaneously detecting multiple functional structural lipids in infant biscuits, wherein the functional structural lipids include any one or more of 1,3-dioleoyl-2-palmitoylglycerol, 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride, and 1-linoleoyl-2-palmitoyl-3-linoleic acid triglyceride. The method uses the aforementioned kit for detection and includes the following steps: (1) After crushing the infant biscuit sample, it was extracted with a mixed extraction solution to obtain a crude extract; (2) The crude extract was purified by solid-phase extraction using purification materials to obtain the test solution; (3) The test solution is detected by a high performance liquid chromatography-dual detector system, and the high performance liquid chromatography separation adopts a gradient elution program; (4) Calculate the content of various functional structural lipids in infant biscuits based on the test results.

[0014] In some methods, after the infant biscuit sample described in step (1) is pulverized and passed through an 80-100 mesh sieve, 1.0 g of sample powder is accurately weighed and placed in a 50 mL centrifuge tube, 10 mL of mixed extraction solution is added, and the mixture is extracted by shaking at 40-50℃ and 150-200 r / min for 20-30 min. The mixture is then centrifuged at 4000-8000 r / min for 5-10 min, and the supernatant is collected. The lower layer residue is extracted twice, and the supernatants are combined to obtain the crude extract.

[0015] In some methods, the solid-phase extraction purification in step (2) includes: first activating the solid-phase extraction column with 5-10 mL of hexane-isopropanol mixture (volume ratio 1:1), then loading the crude extract at a flow rate of 1-2 mL / min, and after the crude extract has completely passed through the column, eluting with 10-15 mL of eluent (hexane-isopropanol mixture, volume ratio 3:2), collecting the eluent, concentrating it to near dryness in a nitrogen blower at 40℃, adding 2 mL of mobile phase to redissolve, and filtering through a 0.22 μm organic filter membrane to obtain the test solution.

[0016] In some methods, the high-performance liquid chromatography-dual detector system described in step (3) includes C 18 The chromatographic column, ultraviolet detector (UV), and evaporative light scattering detector (ELSD) are used; in the gradient elution program, mobile phase A is acetonitrile and mobile phase B is isopropanol.

[0017] In some methods, the gradient elution procedure specifically includes: At 0.00 min, acetonitrile was 88.0% and isopropanol was 12.0%. At 20.00 min, the acetonitrile content was 65.0% and the isopropanol content was 35.0%. At 21.00 min, acetonitrile was 55.0% and isopropanol was 45.0%. At 25.00 min, acetonitrile was 55.0% and isopropanol was 45.0%. At 26.00 min, acetonitrile was 88.0% and isopropanol was 12.0%. At 30.00 min, the acetonitrile content was 88.0% and the isopropanol content was 12.0%.

[0018] Furthermore, the ultraviolet detector is connected to the outlet of the chromatographic column, and the evaporative light scattering detector is connected to the outlet of the ultraviolet detector to achieve tandem detection.

[0019] The high-performance liquid chromatography-dual detector system of this invention employs a specific combination of chromatographic column and detector. Specifically, a C18 column is used to separate three C52-structured lipids: OPO, OPL, and LPL; a UV detector is connected to the column outlet for measuring high-concentration target analytes; and an evaporative light scattering detector is connected to the UV detector outlet for measuring low-concentration target analytes. This tandem detection method enables simultaneous detection of target analytes at different concentrations in the same eluent, effectively solving the concentration matching problem when simultaneously detecting multiple C52-structured lipids.

[0020] Furthermore, the ultraviolet detector has a detection wavelength of 210 nm and is used to determine target substances with a concentration ≥500 μg / mL; the evaporative light scattering detector has a drift tube temperature of 40~60℃, a nitrogen pressure of 3.5~4.0 bar, and a gain value of 1~2 and is used to determine target substances with a concentration <500 μg / mL.

[0021] In some methods, the quantitative calculation in step (4) includes: preparing mixed standard solutions of OPO, OPL and LPL with different concentrations ranging from 5.0 to 10000 μg / mL, detecting them with an ultraviolet detector and an evaporative light scattering detector respectively, plotting a standard curve with the target concentration as the abscissa and the peak area as the ordinate, substituting the peak area of ​​the sample test solution into the standard curve equation of the corresponding concentration level, and calculating the content of OPO, OPL and LPL in the sample.

[0022] In another aspect, the present invention provides the application of the above-mentioned kit and method in the simultaneous detection of the content of three functional structural lipids in infant biscuits, wherein the functional structural lipids include 1,3-dioleoyl-2-palmitoylglycerol, 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride and 1-linoleoyl-2-palmitoyl-3-linoleic acid triglyceride.

[0023] In another aspect, the present invention provides the application of the above-mentioned purification material in the simultaneous detection of multiple functional structural lipids in infant biscuits. The purification material is used for solid-phase extraction purification of the pretreatment of infant biscuit samples. The functional structural lipids include any one or more of 1,3-dioleoyl-2-palmitoylglycerol, 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride, and 1-linoleoyl-2-palmitoyl-3-linoleic acid triglyceride.

[0024] The beneficial effects of this invention include: 1. This invention integrates three C52-structured lipids—OPO, OPL, and LPL—that are difficult to detect simultaneously into a single processing method. By using a C18 chromatographic column and an optimized gradient elution program, it can achieve precise separation of the three C52-structured lipids with a resolution of ≥1.5, avoiding the measurement deviation caused by peak overlap in existing gas chromatography methods. At the same time, compared with gas chromatography, it eliminates the need for saponification and methyl esterification pretreatment, simplifying the operation process and shortening the analysis time.

[0025] 2. The purification material of this invention uses a modified graphene / silica gel composite adsorbent, which combines the high specific surface area of ​​graphene with the adsorption stability of silica gel. By introducing hydrophobic groups through modification, it can selectively adsorb impurities such as proteins, phospholipids, and starch in the sample, while avoiding the adsorption of target substances. The impurity removal rate is high, improving the detection accuracy. The recovery rate of target substances is stable at 94.0%~102.5%, and the relative standard deviation (RSD) is ≤6.0%.

[0026] 3. This invention uses an ultraviolet detector and an evaporative light scattering detector in series to measure high-concentration and low-concentration target substances respectively. This avoids detector saturation for high-concentration target substances and improves the detection sensitivity for low-concentration target substances. The detection limit and quantitation limit are better than existing methods, which can meet the comprehensive determination needs of trace to large amounts of functional structural lipids in infant biscuits.

[0027] 4. The pretreatment process of this invention is innovative. It uses a self-made n-hexane-isopropanol-methanol-1,4-dioxane mixed extract, which is suitable for starch-containing matrices in infant biscuits. Compared with traditional extracts, it has higher fat extraction efficiency, reduces the loss of target substances, and improves extraction efficiency by more than 10%.

[0028] 5. This invention is applicable to starch-containing complementary food products such as infant biscuits, and has wide applicability. It is also applicable to food bases that do not contain starch.

[0029] 6. This invention is simple to operate, efficient, and highly practical. The entire measurement process does not require complex equipment, the pretreatment steps are simplified, the analysis efficiency is high, and samples can be processed in batches. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Detection of OPO, OPL, and LPL in infant biscuits This embodiment uses an infant biscuit sample as an example. The infant biscuit in this embodiment is a commercially available infant teething biscuit (Infant Standard GB10769).

[0032] 1. Preparation of mixed extract Measure 500 mL of n-hexane, 300 mL of isopropanol, 100 mL of methanol and 20 mL of 1,4-dioxane, respectively, mix them well, and obtain a mixed extract with a volume ratio of n-hexane-isopropanol-methanol-1,4-dioxane of 5:3:1:0.2.

[0033] 2. Preparation of modified graphene / silica composite adsorbent (1) Preparation of primary product of graphene / silica composite adsorbent: Weigh 0.4 g of graphene oxide material and add 100 mL of distilled water. Vortex for 4 h to form a suspension. Slowly add 200 mL of anhydrous ethanol solution and stir for 20 min. Slowly add ammonia solution to adjust the pH to 9.5. Slowly add 20 mL of tetramethoxysilane and stir at 50 °C for 12 h to form primary product particles of graphene / silica composite adsorbent. Filter and wash with anhydrous ethanol 3-5 times. Vacuum dry at 60 °C to constant weight to obtain primary product of modified graphene / silica composite adsorbent.

[0034] (2) Preparation of modified graphene / silica composite adsorbent: Take 5 g of the above primary product, add 50 mL of toluene and 1 mL of 3-aminopropyltrimethoxysilane, reflux at 105℃ for 8 h, cool and filter, and vacuum dry at 60℃ to constant weight to obtain modified graphene / silica composite adsorbent.

[0035] 3. Preparation of solid-phase extraction column A solid-phase extraction column was filled with 200 mg of the modified graphene / silica gel composite adsorbent described above, and prepared for use.

[0036] 4. Sample pretreatment Take a sample of infant biscuits, crush it, pass it through an 80-mesh sieve, mix it well, accurately weigh 1.0 g of sample powder into a 50 mL centrifuge tube, add 10 mL of mixed extraction solution (hexane-isopropanol-methanol-1,4-dioxane volume ratio 5:3:1:0.2), place it in a constant temperature water bath shaker, and extract by shaking at 45℃ and 180 r / min for 20 min. Take it out and centrifuge at 4℃ and 4000 r / min for 8 min, and take the supernatant; repeat the extraction twice for the lower layer residue, combine the supernatants to obtain the crude extract.

[0037] 5. Purification The solid-phase extraction column was activated with 8 mL of a hexane-isopropanol mixture (1:1), and the crude extract was loaded onto the column. The flow rate was controlled at 1 mL / min. The column was eluted with 12 mL of elution buffer (hexane-isopropanol 3:2), and the eluent was collected. The eluent was concentrated to near dryness by nitrogen blowing at 40 °C, redissolved with 2.00 mL of mobile phase, and filtered through a 0.22 μm organic filter membrane to obtain the test solution.

[0038] 6. High-performance liquid chromatography separation A C18 column (4.6 mm × 250 mm, 5 μm) was used at a column temperature of 35 °C. The mobile phase was acetonitrile-isopropanol, and the gradient elution program was as shown in Table 1. The flow rate was 1.0 mL / min and the injection volume was 20 μL.

[0039] Table 1 Liquid Phase Gradient Elution Program

[0040] 7. Determination using dual detectors The UV detector was set at a wavelength of 210 nm and a cell temperature of 30 °C. The ELSD detector was set at a drift tube temperature of 50 °C, a nitrogen pressure of 3.8 bar, and a gain of 2. High and low concentrations of the target analyte were measured respectively.

[0041] 8. Preparation of standard solutions and plotting of standard curves Weigh 10 mg (accurate to 0.1 mg) of OPO, OPL and LPL standards into small beakers, dissolve them with the mobile phase and transfer them to 10 mL volumetric flasks, dilute to the mark, mix well and obtain the standard stock solution (1000 μg / mL).

[0042] Mixed standard solutions of OPO, OPL, and LPL were prepared at concentrations of 5.0, 25.0, 100.0, 250.0, 500.0, 1000.0, 2500.0, 5000.0, and 10000 μg / mL. The standard working solutions were injected in ascending order of concentration, and measurements were performed under the aforementioned instrument conditions. A linear regression was performed with the standard working solution concentration as the x-axis and the peak area of ​​the target compound as the y-axis. The linear equation and correlation coefficient R0 were obtained. 2 The detection limit and linear range are shown in Table 2.

[0043] Table 2. Three lipid regression equations, correlation coefficients, limits of detection, and limits of quantification.

[0044] As shown in Table 2, the correlation coefficients of the linear equations for the three lipids were all greater than 0.998; the limits of detection were 0.0030-0.30 mg / g; and the limits of quantitation were 0.010-1.0 mg / g.

[0045] 9. Methodological Validation A biscuit sample was selected and measured in parallel 6 times according to the above method. Three-level spiked recovery tests were performed to examine the recovery rate and precision. The results are shown in Table 3.

[0046] Table 3 Spike recovery and precision tests in biscuit samples

[0047] ND indicates not detected As shown in Table 3, the recovery rates of the three functional structural lipids ranged from 94.1% to 102.5%, and the RSD% ranged from 1.7% to 5.1%, indicating that the recovery rate and precision of this embodiment meet the detection requirements.

[0048] Example 2: Detection of OPO, OPL, and LPL in infant cookies This embodiment uses infant cookies as an example. In this embodiment, infant cookies are children's edible cookies (ordinary modified snacks).

[0049] 1. Preparation of mixed extract Measure 400 mL of n-hexane, 400 mL of isopropanol, 100 mL of methanol and 20 mL of 1,4-dioxane, respectively, mix them well, and obtain a mixed extract with a volume ratio of n-hexane-isopropanol-methanol-1,4-dioxane of 4:4:1:0.2.

[0050] 2. Preparation of modified graphene / silica composite adsorbent Same as Example 1.

[0051] 3. Preparation of solid-phase extraction column Same as Example 1.

[0052] 4. Sample pretreatment Take infant cookie samples, pulverize them, pass them through a 100-mesh sieve, mix them well, accurately weigh 1.0 g of sample powder and place it in a 50 mL centrifuge tube, add 10 mL of mixed extraction solution (hexane-isopropanol-methanol-1,4-dioxane volume ratio 4:4:1:0.2), place it in a constant temperature water bath shaker, and extract by shaking at 45℃ and 200 r / min for 30 min. Take it out and centrifuge at 4℃ and 4000 r / min for 10 min, and take the supernatant; repeat the extraction twice for the lower layer residue, combine the supernatants to obtain the crude extract.

[0053] 5. Purification The solid-phase extraction column was activated with 5 mL of a hexane-isopropanol mixture (1:1), and the crude extract was loaded onto the column. The flow rate was controlled at 1 mL / min. The column was eluted with 10 mL of elution buffer (hexane-isopropanol 3:2), and the eluent was collected. The eluent was concentrated to near dryness by nitrogen blowing at 40 °C, redissolved with 2.00 mL of mobile phase, and filtered through a 0.22 μm organic filter membrane to obtain the test solution.

[0054] 6. High-performance liquid chromatography separation A C18 column (4.6 mm × 250 mm, 5 μm) was used at a column temperature of 30 °C. The mobile phase was acetonitrile-isopropanol, and the gradient elution program was as shown in Table 1. The flow rate was 1.0 mL / min and the injection volume was 20 μL.

[0055] 7. Determination using dual detectors The UV detector was set at a wavelength of 210 nm and a cell temperature of 30 °C. The ELSD detector was set at a drift tube temperature of 50 °C, a nitrogen pressure of 3.5 bar, and a gain of 1. High and low concentrations of the target analyte were measured respectively.

[0056] 8. Preparation of standard solutions and plotting of standard curves Same as Example 1.

[0057] 9. Methodological Validation A cookie sample was selected and measured in parallel 6 times according to the above method. Three-level spiked recovery tests were performed to examine the recovery rate and precision. The results are shown in Table 4.

[0058] Table 4 Spiked recovery and precision tests in cookie samples

[0059] As shown in Table 4, the recovery rates of the three functional structural lipids ranged from 94.2% to 98.2%, and the RSD% ranged from 1.1% to 5.5%, indicating that the recovery rate and precision of this embodiment meet the detection requirements.

[0060] Example 3: Comprehensive Comparison of the Method of the Present Invention with Existing Technologies and Methods This embodiment aims to systematically evaluate the advantages of the method of the present invention compared with the prior art (patent CN 121453956 A) and conventional methods (GB 30604-2015), using infant biscuits as samples.

[0061] 1. Comparison with conventional methods This embodiment compares the effects of the method of the present invention and conventional methods on the detection results of OPO, OPL, and LPL in infant biscuit samples. It should be noted that the three functional structural lipids detected in this invention require determination according to GB30604-2015 in conventional methods. This standard only applies to pure OPO nutritional fortifiers using gas chromatography and cannot distinguish between OPL and LPL, and is not applicable to final products such as infant biscuits.

[0062] Samples of infant biscuits from the same batch were tested using the method of Example 1 of this invention and the conventional method described above. The test results and operational efficiency were compared, and the results are shown in Table 5.

[0063] Table 5 Comparison between this method and conventional methods

[0064] Experimental results show that the method of the present invention significantly improves detection efficiency, reduces time and reagent consumption, and can simultaneously detect three functional structural lipids with good recovery rate and precision. Conventional methods are not applicable to infant biscuit samples, and cannot simultaneously detect OPL and LPL.

[0065] 2. Comparison with existing gas chromatography methods This embodiment compares the results of the method of the present invention with those of the method described in patent CN 121453956 A in detecting functional structural lipids in infant biscuit samples. The results are shown in Table 6.

[0066] Table 6 Comparison between this method and the patented method

[0067] As shown in Table 6, compared with the method described in patent CN 121453956 A, the method of the present invention can detect more types of substances, and adds LPL; the purification effect is better, and it is applicable to complementary food products such as infant biscuits containing starch, which has significant advantages.

[0068] This invention's method is applicable to starch-containing complementary food products such as infant biscuits. Considering that infant biscuit matrices contain a large amount of starch, and that starch molecules form a complex spatial network structure through hydrogen bonds and hydrophobic interactions, exhibiting strong encapsulation and adsorption effects on functional structural lipids, and that biscuits also contain various interfering components such as proteins and carbohydrates, the extraction, purification, and detection processes pose significant challenges. Therefore, to fully verify the effectiveness and superiority of this invention's method under complex matrix conditions, all comparative examples below use infant biscuits as representative matrices for evaluation.

[0069] Comparative Example 1: Comparison of the effects of different extracts and compositions This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the effects of different extracts and compositions on the detection results of OPO, OPL, and LPL in infant biscuit samples. Since the three lipids have relatively small differences in polarity, and the starch network structure in infant biscuits has a lipid-encapsulating effect, the extract needs to have good permeability and solubility. Formula A is petroleum ether (referring to the commonly used extraction solvent in the first method of GB 5009.6-2016); Formula B is n-hexane + isopropanol (volume ratio 3:2, referring to the classic binary system of the Hara-Radin method); Formula C is n-hexane + isopropanol + methanol (volume ratio 5:3:1, the ternary system after removing 1,4-dioxane in this invention); Formula D is n-hexane + isopropanol + methanol + 1,4-dioxane (volume ratio 5:3:1:0.2, the quaternary system of this invention); Formula E is n-hexane + isopropanol + methanol + tetrahydrofuran (THF) (volume ratio 5:3:1:0.2, the quaternary system in which THF replaces 1,4-dioxane). The results are shown in Table 7.

[0070] Table 7. Effects of different extraction solutions on the recovery rate of three lipids and the total fat extraction rate.

[0071] As shown in Table 7, traditional petroleum ether (Formula A), as a single non-polar solvent, has extremely poor penetration ability for lipids encapsulated in the starch network structure, resulting in the lowest extraction efficiency. The Hara-Radin binary system (Formula B), after introducing isopropanol, increases solvent polarity, enhances lipid solubility, and improves extraction efficiency to some extent, but still cannot effectively disrupt the starch network structure. The ternary system (Formula C) further adds methanol, enhancing polarity regulation and significantly improving the extraction efficiency for relatively polar lipids such as LPL. However, methanol has limited penetrating and disrupting effects on the starch structure and easily causes partial starch gelatinization, increasing the dissolution of impurities.

[0072] Formulation E uses tetrahydrofuran instead of the 1,4-dioxane of this invention. Although both are cyclic ether solvents, THF has a five-membered ring structure containing only one oxygen atom, and its hydrogen bond acceptance and steric hindrance are significantly different from those of 1,4-dioxane. THF's recognition of hydrogen bonds between starch molecules is not precise enough, and while disrupting the starch network structure, it easily causes local over-penetration, leading to increased starch gelatinization and increased dissolution of impurities. At the same time, THF's selectivity for lipid dissolution is lower than that of 1,4-dioxane, causing the target substance and impurities to dissolve simultaneously, and the recovery rates of the three lipids are significantly lower than those of this invention.

[0073] This invention's quaternary system (Formula D) introduces 1,4-dioxane into methanol. 1,4-dioxane, as a six-membered ring ether solvent, contains two oxygen atoms and possesses good permeability and moderate hydrogen bond accepting capacity. It can effectively disrupt the hydrogen bond network between starch molecules, promoting the precise release of lipids from the starch matrix. Simultaneously, 1,4-dioxane can inhibit the dissolution of impurities during starch gelatinization, reducing matrix interference. Methanol and 1,4-dioxane exhibit a positive synergistic effect, with an average recovery rate of over 94% for the three lipids, resulting in an overall extraction efficiency improvement of over 10%.

[0074] In summary, the hexane-isopropanol-methanol-1,4-dioxane quaternary system of this invention is not only superior to traditional binary and ternary systems in terms of the number of solvents, but also has specificity in the selection of solvent types. The six-membered ring dioxygen atom structure of 1,4-dioxane is the key to achieving precise destruction of starch network and selective release of lipids, and cannot be simply replaced by other cyclic ether solvents.

[0075] Comparative Example 2: Comparison of the effects of different purification materials This comparative example examines the effects of different purification materials on the detection results of OPO, OPL, and LPL in infant biscuit samples under the optimal conditions of the method of this invention (Example 1) in all other conditions. Infant biscuit matrices are complex, containing various interfering components such as proteins, phospholipids, and pigments in addition to starch. These interfering components are extracted simultaneously with the target analytes during the extraction process. Poor purification will severely affect the accuracy of chromatographic separation and detection. Therefore, the selective adsorption capacity of the purification material is crucial.

[0076] Material A is the modified graphene / silica gel composite adsorbent of this invention (200 mg), which is obtained by reacting graphene oxide with tetramethoxysilane to form a composite, followed by amino modification with 3-aminopropyltrimethoxysilane; Material B is conventional silica gel (200 mg); Material C is a C18 adsorbent (200 mg); Material D is neutral alumina (200 mg); Material E is untreated; Material F is unmodified graphene / silica gel composite adsorbent (200 mg), prepared in the same way as step (1) of this invention, omitting the 3-aminopropyltrimethoxysilane modification step; Material G is octadecyl-modified graphene / silica gel composite adsorbent (200 mg), prepared in the same way as this invention, but modified with octadecyltrimethoxysilane instead of 3-aminopropyltrimethoxysilane. The results are shown in Table 8.

[0077] Table 8. Effects of different purification materials on the recovery rates of three lipids.

[0078] As shown in Table 8, compared with the untreated sample (Material E), the recovery rates of all target analytes were significantly improved after using the purified materials, indicating that the purification step plays an important role in removing matrix interference and improving detection accuracy. Compared with the traditional silica gel adsorbent (Material B), the modified graphene / silica gel composite adsorbent of this invention (Material A) showed an increase in the recovery rates of OPO, OPL, and LPL of 12.3%, 13.7%, and 18.5%, respectively, with a significant reduction in matrix interference and a marked improvement in chromatographic peak shape. This is mainly due to the combination of the high specific surface area of ​​graphene and the adsorption stability of silica gel in the material of this invention. The introduction of hydrophobic groups through amino modification allows for the selective adsorption of polar impurities such as proteins, phospholipids, and starch, while avoiding non-specific adsorption of target lipids. Compared with the C18 adsorbent (Material C), the recovery rates of the material of this invention increased by 9.0%, 11.0%, and 15.8%, respectively, indicating that although C18 has a certain retention capacity for lipids, its selective removal capacity for impurities is insufficient and it is prone to causing loss of target analytes. Compared with neutral alumina (material D), the recovery rates of the materials in this invention are increased by 16.2%, 18.4%, and 23.2%, respectively, indicating that alumina has a strong adsorption effect on lipids, resulting in serious loss of the target substance, and is not effective in removing impurities such as starch.

[0079] While the unmodified graphene / silica gel composite adsorbent (material F) retains the high specific surface area of ​​graphene and the framework structure of silica gel, it lacks the selective recognition ability of amino hydrophobic groups. The unmodified composite material has a hydrophilic surface, resulting in insufficient adsorption selectivity for polar impurities such as proteins and phospholipids. Some target lipids are non-specifically adsorbed. The recoveries of OPO, OPL, and LPL are 88.7%, 86.4%, and 84.2%, respectively, representing reductions of 5.9%, 7.8%, and 11.1% compared to this invention, with a moderate degree of matrix interference. These results demonstrate that the 3-aminopropyltrimethoxysilane modification step is crucial for improving the selectivity of the purification material and achieving precise impurity removal.

[0080] The octadecyl-modified graphene / silica composite adsorbent (material G) introduces a long-chain hydrophobic group (C18), which, while showing good removal efficiency for polar impurities (high impurity removal rate), results in excessively strong hydrophobic interactions between the C18 chain and the target lipid molecules, leading to non-specific adsorption and elution difficulties of the target analyte on the column. The recoveries of OPO, OPL, and LPL were 83.2%, 81.5%, and 78.9%, respectively, representing reductions of 11.4%, 12.7%, and 16.4% compared to the present invention. Although material G achieves a high impurity removal rate, its non-specific adsorption of the target lipid is also enhanced, resulting in significant loss of the target analyte. These results indicate that the length and polarity of the amino-modified hydrophobic group must be precisely matched to the target lipid molecule: the short-chain amino hydrophobic group provided by 3-aminopropyltrimethoxysilane can selectively recognize polar impurities without excessively retaining non-polar lipids, making it the optimal choice for balancing high recovery and high purification efficiency.

[0081] In summary, the modified graphene / silica gel composite adsorbent of this invention exhibits significant advantages in both recovery rate and purification effect. Compared with traditional silica gel adsorbents, the impurity removal rate is increased by more than 20%, and the target analyte recovery rate is increased by 12%~18%. Compared with unmodified composite materials, the modification step increases the recovery rate by 6%~11%, demonstrating the necessity of modification. Compared with other hydrophobic group modifications (octadecyl), amino modification achieves the optimal balance between target analyte retention and impurity removal, providing a specific material solution for the efficient purification of functional structural lipids in complex matrices such as infant biscuits.

[0082] Comparative Example 3: Comparison of the effects of different chromatographic separation conditions This comparative example, under the optimal conditions of the method of the present invention (Example 1) in all other aspects, systematically investigated the effects of different chromatographic separation conditions on the separation of three C52-structured lipids (OPO, OPL, and LPL) in infant biscuit samples. Since OPO, OPL, and LPL are all C52-structured lipids with similar molecular weights and minimal polarity differences, differing only in the attachment positions of fatty acids on the glycerol backbone, the selection of chromatographic separation conditions is crucial for achieving baseline separation of these three lipids.

[0083] Condition A is the method of this invention (C18 column, acetonitrile-isopropanol gradient elution, column temperature 35℃); Condition B is isocratic elution conditions (C18 column, acetonitrile-isopropanol volume ratio 70:30 isocratic elution, column temperature 35℃); Condition C is a conventional reversed-phase C18 column with a methanol-water system (C18 column, methanol-water volume ratio 90:10 isocratic elution, column temperature 35℃); Condition D is the gas chromatography separation conditions described in patent CN 121453956 A (CP-TAP CB capillary column, programmed temperature rise, FID detector); Condition E is supercritical fluid chromatography conditions (C18 column, supercritical CO2-methanol gradient elution, column temperature 40℃). The results are shown in Table 9.

[0084] Table 9. Effects of different chromatographic separation conditions on the separation efficiency of three lipids.

[0085] As shown in Table 9, under isocratic elution conditions (condition B) with a fixed acetonitrile-isopropanol volume ratio of 70:30, the three C52 lipid structures elute almost simultaneously with a retention time of 12.45 min due to their minimal polarity differences. This resulted in complete insemination, with severe peak overlap, making quantitative analysis impossible. The methanol-water system (condition C), a commonly used mobile phase in conventional reversed-phase chromatography, also failed to separate the three lipids due to their similar hydrophobicity. Furthermore, the methanol-water system has limited solubility for non-polar lipids, easily leading to column blockage and peak shape deterioration.

[0086] The gas chromatography method (condition D) described in patent CN 121453956 A uses a CP-TAP CB capillary column combined with programmed temperature rise, which can effectively separate OPO and OPL. However, it has the following limitations: First, it only covers OPO and OPL and cannot detect LPL; second, it is only suitable for infant formula milk powder as the matrix. For complementary food products such as infant biscuits containing starch, it is not suitable due to the fat encapsulation effect of the starch network structure and the risk of lipid thermal degradation caused by the high temperature operation (up to 350℃) of GC; third, it requires alkaline hydrolysis pretreatment, which is relatively complicated.

[0087] The method of this invention (Condition A) employs an optimized acetonitrile-isopropanol gradient elution program. By precisely controlling the rate of change of mobile phase polarity (acetonitrile 88.0%, isopropanol 12.0% at 0.00 min, gradually transitioning to acetonitrile 55.0%, isopropanol 45.0% at 21.00 min), the precise separation of three C52-structured lipids is achieved. Among them, OPO (1,3-dioleoyl-2-palmitoylglycerol triglyceride), containing two oleic acid residues (C18:1), has relatively strong hydrophobicity and is eluted first; OPL (1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride), containing one oleic acid and one linoleic acid residue (C18:2), has moderate hydrophobicity; LPL (1-linoleoyl-2-palmitoyl-3-linoleic acid triglyceride), containing two linoleic acid residues, has the weakest hydrophobicity and is eluted last. The pairwise resolutions of the three are as follows: OPO to OPL 2.12, OPL to LPL 1.87, and OPO to LPL 3.85, all ≥1.5, meeting the baseline separation standard. The chromatographic peaks are sharp and symmetrical, the analysis time is moderate (30 min), and no derivatization is required. The molecular structure information of triglycerides is completely preserved, and isomer identification can be performed directly.

[0088] Meanwhile, the separation degree between OPO and LPL reached 3.85, which is the highest among the three lipid pairs. This further verifies that the gradient elution program of this invention has excellent separation selectivity for C52 structural lipid isomers. This separation degree data fully demonstrates that the method of this invention can not only distinguish between adjacent peaks of OPL and LPL, but also clearly distinguish between OPO and LPL with the greatest polarity difference, fundamentally solving the chromatographic separation problem of simultaneous detection of three structural lipids.

[0089] In summary, the optimized gradient elution program of this invention has significant advantages in the separation of three C52 structural lipids. Compared with existing isocratic elution, methanol-water system and supercritical fluid chromatography conditions, the resolution is improved from 0 to ≥1.5. Compared with gas chromatography conditions (only two can be separated), the addition of LPL detection and the realization of simultaneous baseline separation of three types of lipids have achieved simultaneous baseline separation of OPO, OPL and LPL for the first time, providing a reliable chromatographic separation basis for the accurate quantification of multiple functional structural lipids in infant biscuits.

[0090] Comparative Example 4: Performance Comparison of Different Detector Configurations This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the effects of different detector configurations on the detection of three functional structural lipids, OPO, OPL, and LPL, in infant biscuit samples. Since the amounts of the three C52 structural lipids added to infant biscuits differ significantly (OPO is usually added in constant amounts, OPL in medium amounts, and LPL in trace amounts), and the concentration range of the three in actual samples can reach 3-4 orders of magnitude, the choice of detector configuration directly determines whether the method can simultaneously meet the dual requirements of accurate quantification at high concentrations and sensitive detection at low concentrations.

[0091] Configuration A consists of a UV detector and an evaporative light scattering detector connected in series. The UV detector is connected to the column outlet, and the ELSD detector is connected to the UV detector outlet. Intelligent switching is achieved by setting a concentration threshold of 500 μg / mL. High concentrations are quantified using UV, and low concentrations using ELSD. The same flow path, the same peak, and identical retention times are maintained, allowing a single injection to cover 5–10,000 μg / mL. Configuration B uses a single UV detector with a detection wavelength of 210 nm, a linear range of 500–10,000 μg / mL, and a detection limit of 0.30 mg / g. Configuration C uses a single evaporative light scattering detector with a drift tube temperature of 50°C, a nitrogen pressure of 3.8 bar, a gain of 2, a linear range of 5–500 μg / mL, and a detection limit of 0.0030 mg / g. However, it exhibits non-linear response to high-concentration samples, with a quantification deviation greater than 15%. Configuration D uses a single flame ionization detector, as described in patent CN 121453956. Method A requires alkaline hydrolysis pretreatment and operates at a maximum temperature of 350℃. Configuration E consists of a parallel connection of a UV detector and an evaporative light scattering detector, with a split flow path and a fixed split ratio. This leads to mismatched peak areas, and fluctuations in the split ratio cause quantitative deviations. The results are shown in Table 10.

[0092] Table 10. Effects of different detector configurations on the detection performance of three types of lipids

[0093] Table 10 shows that a single UV detector (configuration B) exhibits good linearity for high-concentration target analytes, but has a high detection limit, failing to detect trace amounts of LPL; a single evaporative light scattering detector (configuration C) offers high sensitivity at low concentrations, but its response to high-concentration samples is nonlinear; a single flame ionization detector (configuration D) requires alkaline hydrolysis pretreatment, which does not damage the triglyceride structure, but high-temperature operation (up to 350℃) poses a risk of degradation to thermally unstable lipids. The parallel configuration (configuration E) results in mismatched peak areas due to current splitting, leading to significant quantitative deviation.

[0094] The tandem configuration of this invention (Configuration A) covers three orders of magnitude with a single injection, perfectly adapting to the detection needs of three lipids with huge differences in concentration: OPO (constant), OPL (medium), and LPL (micro).

[0095] Verification was conducted using actual infant biscuit samples: One sample contained 12.5 mg / g OPO, 0.85 mg / g OPL, and 0.12 mg / g LPL. Using a single UV analyzer, OPL was below the limit of quantification, and LPL was below the limit of detection. Using a single ELSD analyzer, the OPO saturation deviation was 18.7%. Using a parallel configuration, the RSD was greater than 8%. Using the tandem configuration of this invention, OPO was automatically quantified using UV (RSD 2.1%), and OPL and LPL were automatically quantified using ELSD (RSD 2.9% and 3.9%, respectively), yielding accurate results for all three lipids.

[0096] In summary, the UV-ELSD dual detector tandem configuration of this invention extends the concentration range by one order of magnitude, eliminates the peak area mismatch and shunting optimization problems of parallel configuration, and achieves for the first time the simultaneous and accurate determination of trace to constant amounts of functional structural lipids in infant biscuits.

[0097] Comparative Example 5: Comparison of the effects of different SPE eluent formulations This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, investigates the effects of different SPE eluent formulations on the recovery rates and purification effects of OPO, OPL, and LPL in infant biscuit samples. The polarity of the eluent directly affects the desorption efficiency of the target analyte from the modified graphene / silica composite adsorbent and the degree of co-elution of impurities.

[0098] Formula A is a hexane-isopropanol mixture (volume ratio 3:2); Formula B is hexane-isopropanol (volume ratio 4:1); Formula C is hexane-isopropanol (volume ratio 1:1); and Formula D is hexane-isopropanol (volume ratio 2:3). The results are shown in Table 11.

[0099] Table 11 Effects of different SPE eluent formulations on the recovery rate and purification effect of three lipids

[0100] Table 11 shows that formulation B (4:1) has a high hexane ratio, resulting in weak polarity and insufficient elution driving force for the three lipids, leading to low recoveries (82.7%~88.6%). Formulation C (1:1) has increased polarity and improved recovery, but increased co-elution of impurities and higher matrix interference. Formulation D (2:3) has an excessively high isopropanol ratio; although it has the highest recovery, its selectivity is reduced, a large number of polar impurities are simultaneously eluted, resulting in deteriorated peak shapes and significant matrix interference.

[0101] Formulation A (3:2) of this invention achieves an optimal balance between target analyte recovery (94.2%~95.3%) and impurity removal rate, with good chromatographic peak shape and low matrix interference.

[0102] In summary, the hexane-isopropanol volume ratio 3:2 eluent formulation of this invention balances target analyte elution efficiency and purification selectivity, providing optimized elution conditions for purification of modified graphene / silica composite adsorbent SPE.

[0103] Comparative Example 6: Comparison of the effects of different extraction times This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the effects of different extraction times on the recovery rates of OPO, OPL, and LPL and the operational efficiency in infant biscuit samples. With a fixed total solvent volume of 30 mL, the effect of the number of extractions on the completeness of lipid release from the starch network structure was compared.

[0104] Method A involves three extractions, with 10 mL of the mixed extract added each time, followed by shaking for 20 min and centrifugation, and the supernatants from the three extractions are combined. Method B is a one-step extraction, with 30 mL of the mixed extract added at once, followed by shaking for 60 min and centrifugation. Method C is a two-step extraction, with 15 mL of the mixed extract added each time, followed by shaking for 30 min and centrifugation, and the supernatants from the two extractions are combined. The results are shown in Table 12.

[0105] Table 12 Effects of different extraction times on the recovery rate and operational efficiency of the three lipids

[0106] As shown in Table 12, although the one-step extraction (Method B) is the simplest to operate, the penetration depth of the large amount of solvent in a single extraction is limited, and the high volume of solvent leads to a decrease in oscillation efficiency. A large amount of lipids are still trapped inside the starch and cannot be released. The recovery rates of the three lipids are all below 85%, and the total fat extraction rate is only 80.6%. The two-step extraction (Method C) maintains a certain concentration gradient driving force by changing the solvent in stages, and the recovery rate is improved. However, the concentration of residual lipids in the matrix has decreased in the second extraction, and the extraction driving force is weakened. The recovery rates of OPO, OPL, and LPL are still 3.0 to 7.9 percentage points lower than those of the present invention.

[0107] This invention employs a three-stage extraction method (Method A) that maintains a high concentration gradient driving force in each extraction by replacing the solvent several times, ensuring the full release of lipids encapsulated in the starch network structure. Simultaneously, the solvent volume is moderate per extraction, resulting in high oscillation efficiency and thorough separation of residue and supernatant after centrifugation. The overall recovery rate after combining the three extractions exceeds 94%. The operation steps are standardized, have good repeatability, and are simple in operation. The extraction time for a single extraction is comparable to that of a one-stage extraction, while significantly improving the overall extraction efficiency.

[0108] In summary, the three-stage extraction method of this invention, under the condition of fixed total solvent usage, maintains the concentration gradient driving force through multiple extractions, which significantly improves the release efficiency of starch-encapsulated lipids, and provides optimized operating parameters for the efficient extraction of functional structural lipids from complex starch-containing matrices such as infant biscuits.

[0109] Comparative Example 7: Comparison of the effects of different extraction-purification combinations This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, examines the effects of the combination order and method of extraction and purification on the recovery rates and operational efficiency of OPO, OPL, and LPL in infant biscuit samples. The combination method of extraction and purification directly affects the complexity of the pretreatment process, solvent consumption, and batch processing capacity.

[0110] In this invention, process A involves extraction followed by purification, with the three extractions combined and then subjected to SPE purification. Process B involves stepwise purification, with SPE purification performed separately after each extraction, and the eluents collected after each purification are combined. Process C involves simultaneous extraction and purification, with the SPE column placed in the extraction system and the extract circulating through the SPE column. The results are shown in Table 13.

[0111] Table 13 Effects of different extraction-purification combinations on the recovery rate and operational efficiency of three lipids

[0112] As shown in Table 13, the stepwise purification (process B) requires separate SPE column activation, sample loading, elution, and collection after each extraction. The amount of SPE column used increases by 3 times, the consumption of organic solvents increases by 60%, and the operation time doubles. The operation is complicated. Although the recovery rate is close to that of the present invention (OPO 93.8%, OPL 91.5%, LPL 89.2%), the cost is high and it is not suitable for batch sample detection.

[0113] The simultaneous extraction-purification process (Process C) involves recycling the SPE column within the extraction system to simplify the operation, but it suffers from several problems: First, under oscillation conditions, the SPE adsorbent particles are prone to breakage and loss, leading to the loss of purification material and sample contamination. Second, the adsorbent selectivity gradually decreases during multiple cycles, reducing the impurity removal rate, and the target analyte competes with impurities for adsorption. Third, the circulation flow rate is difficult to control, resulting in some target analytes being adsorbed before full extraction, affecting release efficiency. The recovery rates of the three lipids were significantly lower than those of this invention (OPO decreased by 6.2%, OPL by 8.6%, and LPL by 13.0%), with moderate operational complexity.

[0114] The present invention employs a combined extraction and purification method (process A), which combines three extractions and performs SPE purification in a unified manner. This method ensures the concentration gradient driving force and oscillation efficiency during the extraction process, avoids repetitive purification steps, minimizes organic solvent consumption, and allows the SPE column to achieve the best purification effect with a single use. The operation is simple and has strong batch processing capabilities.

[0115] In summary, the "extraction-merging-unified purification" pretreatment process of this invention achieves an optimal balance between recovery rate, operational efficiency, cost, and batch processing capacity, providing a standardized pretreatment solution for high-throughput detection of functional structural lipids in complex matrices such as infant biscuits.

[0116] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for simultaneously detecting multiple functional structural lipids in infant biscuits, characterized in that, The functional structural lipids include 1,3-dioleoyl-2-palmitoylglycerol, 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride, and 1-linoleoyl-2-palmitoyl-3-linoleic acid triglyceride; the method uses the following kit for detection. The kit includes purification materials and a mixed extraction solution. The purification materials include a modified graphene / silica composite adsorbent, which is obtained by reacting graphene oxide with tetramethoxysilane to form a primary product of graphene / silica composite adsorbent, and then modifying it with 3-aminopropyltrimethoxysilane. The extract is composed of a mixture of n-hexane, isopropanol, methanol, and 1,4-dioxane, wherein the ratio of the n-hexane, isopropanol, methanol, and 1,4-dioxane mixture is 4~6:3~4:1:0.

2. The method includes the following steps: (1) After crushing the infant biscuit sample, it was extracted with a mixed extraction solution to obtain a crude extract; (2) The crude extract was purified by solid-phase extraction using purification materials to obtain the test solution; (3) The test solution is detected by a high performance liquid chromatography-dual detector system, and the high performance liquid chromatography separation adopts a gradient elution program; (4) Calculate the content of various functional structural lipids in infant biscuits based on the test results.

2. The method as described in claim 1, characterized in that, The high-performance liquid chromatography-dual detector system described in step (3) includes C 18 The chromatographic column, ultraviolet detector, and evaporative light scattering detector are used; in the gradient elution program, mobile phase A is acetonitrile and mobile phase B isopropanol.

3. The method as described in claim 2, characterized in that, The gradient elution procedure is as follows: At 0.00 min, acetonitrile was 88.0% and isopropanol was 12.0%. At 20.00 min, the acetonitrile content was 65.0% and the isopropanol content was 35.0%. At 21.00 min, acetonitrile was 55.0% and isopropanol was 45.0%. At 25.00 min, acetonitrile was 55.0% and isopropanol was 45.0%. At 26.00 min, acetonitrile was 88.0% and isopropanol was 12.0%. At 30.00 min, the acetonitrile content was 88.0% and the isopropanol content was 12.0%.

4. The method as described in claim 2, characterized in that, The ultraviolet detector is connected to the outlet of the chromatographic column, and the evaporative light scattering detector is connected to the outlet of the ultraviolet detector to achieve tandem detection.

5. The method as described in claim 2, characterized in that, The ultraviolet detector is used to determine target substances with a concentration ≥500 μg / mL, and the evaporative light scattering detector is used to determine target substances with a concentration <500 μg / mL.

6. The application of the method according to any one of claims 1 to 5 in the simultaneous detection of the content of three functional structural lipids in infant biscuits, characterized in that, The functional structural lipids include 1,3-dioleoyl-2-palmitoyl triglyceride, 1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride, and 1-linoleoyl-2-palmitoyl-3-linoleic acid triglyceride.

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