Method for Accurately Separating and Identifying Oxidized Triglyceride in Frying Oil

Abstract

The present disclosure discloses a method for accurately separating and identifying an oxidized triglyceride in frying oil, and belongs to the technical field of detection. In the present disclosure, the structure of the oxidized triglyceride (ox-TG) in the frying oil is identified by mass spectrometry first. It is found that after frying is conducted for 24 h, the ox-TG mainly includes epoxy ox-TG, hydroxyl ox-TG, and aldehyde ox-TG. Thus, the three types of ox-TG are selected as a template molecule to synthesize a surface molecularly imprinted polymer (SMIPs). Then, a polymer completely matched with the ox-TG template molecule in action site and spatial configuration is synthesized, and the specific ox-TG can be separated by using the SMIPs. According to the present disclosure, OXTG-SMIPs prepared by a molecular imprinting technology has good specificity, stability, and affinity, and accurate separation of the ox-TG in the frying oil can be achieved.

Claims

1. A method for accurately separating and identifying an oxidized triglyceride in frying oil, wherein the method comprises the following steps: (a) preparation of a surface molecularly imprinted polymer: first, grafting an outer layer of a substrate material with a functional monomer; adding a template molecule, namely epoxy ox-TG, hydroxyl ox-TG and/or aldehyde ox-TG; then, after the functional monomer is prepolymerized with the template molecule for 1-2.5 hours, adding an initiator azodiisobutyronitrile and a crosslinking agent ethylene glycol dimethacrylate to fix a complex into a polymer network so as to obtain a crosslinked polymer; and finally, subjecting the template molecule in the crosslinked polymer to elution to obtain an oxidized triglyceride-molecularly imprinted polymer (OXTG-SMIPs), wherein the functional monomer comprises any one of methacrylic acid, acrylamide, and 4-vinylpyridine; (b) preparation of a chromatographic column: loading the OXTG-SMIPs obtained in step (1) into a chromatographic column; (c) separation with the chromatographic column: adding the frying oil to the chromatographic column, and adding an eluting agent for elution so as to separate the oxidized triglyceride from the frying oil; and (d) elution of an eluent: sequentially adding eluting agents to the chromatographic column for eluting the oxidized triglyceride, and conducting separation to obtain epoxy ox-TG, hydroxyl ox-TG and aldehyde ox-TG.

2. The method according to claim 1, wherein in step (a), the substrate material comprises any one or more of silica gel, glucan, and titanium dioxide.

3. The method according to claim 1, wherein in step (a), the functional monomer is the methacrylic acid or the acrylamide.

4. The method according to claim 1, wherein a mass ratio of the substrate material grafted with the functional monomer to the template molecule is 5:1 to 10:1, the use amount of the initiator is 2%-4% (mass/mass) of that of the substrate material, and the use amount of the crosslinking agent is 1.5-2 times (mass/mass) that of the substrate material.

5. The method according to claim 1, wherein in step (a), structural formulas of the hydroxyl ox-TG, the aldehyde ox-TG, and the epoxy ox-TG are as shown in Formula I to Formula III, respectively: ##STR00007## ##STR00008## ##STR00009##

6. The method according to claim 1, wherein when any one of the epoxy ox-TG, the hydroxyl ox-TG, and the aldehyde ox-TG is added, a single-template molecularly imprinted polymer, namely hydroxyl OX-TGMIPs (SMIPs1), aldehyde OX-TGMIPs (SMIPs2), or epoxy OX-TGMIPs (SMIPs3), is prepared.

7. The method according to claim 6, wherein when a single-template molecularly imprinted polymer is prepared, the three types of OXTG-SMIPs, namely the SMIPs1, the SMIPs2, and the SMIPs3, are loaded into the chromatographic column at a mass ratio of 1:1:1 to 1:1:2 in step (b).

8. The method according to claim 1, wherein in step (c), the eluting agent is any one of dimethyl sulfoxide and tetrahydrofuran.

9. The method according to claim 1, wherein in step (c), a mass ratio of the frying oil to the OXTG-SMIPs is 1:20 to 1:25.

10. The method according to claim 1, wherein in step (d), the eluting agents sequentially comprise a mixture of acetic acid and methanol at a ratio of 1:8, a mixture of acetic acid and methanol at a ratio of 1:6, and a mixture of acetic acid and methanol at a ratio of 1:4.

11. The method according to claim 1, wherein the frying oil is edible oil used in families, restaurants, industrial frying, and cooking food.

Description

BRIEF DESCRIPTION OF FIGURES

[0042] FIG. 1A is a mass spectrometry EIC images of main ox-TG when frying oil is separately fried for 0 h.

[0043] FIG. 1B is a mass spectrometry EIC images of main ox-TG when frying oil is separately fried for 12 h.

[0044] FIG. 1C is a mass spectrometry EIC images of main ox-TG when frying oil is separately fried for 24 h.

[0045] FIG. 2 is a structural formulas of hydroxyl ox-TG, aldehyde ox-TG, and epoxy ox-TG.

[0046] FIG. 3 is a schematic diagram showing the synthesis of OXTG-SMIPs.

[0047] FIG. 4 is a diagram showing Fourier infrared spectrum characterization of SiO.sub.2 (a), amino modified SiO.sub.2 (b), SiO.sub.2@acrylamide (c), SMIPs2 (d), and SNIPs (e).

[0048] FIG. 5 is a schematic diagram showing the operation of separating ox-TG from frying oil by means of SMIPs.

[0049] FIG. 6 is a TLC image showing an ox-TG separation effect of a molecular imprinting method in comparison with a conventional silica gel column chromatography method.

[0050] FIG. 7 isa hydrogen nuclear magnetic resonance spectrum image showing an ox-TG separation effect of a molecular imprinting method in comparison with a conventional silica gel column chromatography method.

DETAILED DESCRIPTION

[0051] The present disclosure is further described below in conjunction with examples, but the embodiments of the present disclosure are not limited herein.

[0052] A determination method and calculation formulas of the RSD, detection limit and recovery rate are as follows: the content of ox-TG obtained after elution is determined by Raman spectroscopy, and the RSD (RSD=standard deviation SD/arithmetic mean X), detection limit (S/N=3) and recovery rate (Recovery=m.sub.added/m.sub.recovered) are calculated based on the method.

[0053] The substrate materials including silica gel, glucan and titanium dioxide, methacrylic acid, acrylamide, and 4-vinylpyridine mentioned in the following examples and comparative examples were purchased from Bailingwei Chemical Reagent Co., Ltd..

Example 1: Method for Accurately Separating and Identifying ox-TG in Frying Oil

[0054] (a) First, 5 g of silica gel as a substrate material was weighed and placed in a 500 mL round-bottomed three-mouth flask, 150 mL of anhydrous toluene was added, 30 ml of aminopropyltriethoxysilane and 10 ml of pyridine were slowly added dropwise under magnetic stirring at room temperature, and magnetic stirring was conducted in a water bath under the protection of nitrogen at 95° C. for 24 h to obtain amino modified SiO.sub.2. After drying was completed, 5 g of the amino modified SiO.sub.2 was weighed and added to a 250 mL round-bottomed three-mouth flask, 100 mL of anhydrous toluene was added, magnetic stirring was conducted for 15 min, 8 mL of acryloyl chloride and 5 mL of triethylamine were slowly added dropwise, and after the dropping was completed, magnetic stirring was conducted under the protection of nitrogen for 24 h to obtain SiO.sub.2@acrylamide.

[0055] After drying was completed, 5 g of the SiO.sub.2@acrylamide was weighed, 50 mL of DMSO as a solvent, 0.10 g of azodiisobutyronitrile as an initiator, 8.0 g of ethylene glycol dimethacrylate as a crosslinking agent, and 0.80 g of hydroxyl ox-TG as a template molecule were added for a reaction for 24 h, and then an eluting agent (a mixture of acetic acid and methanol at a ratio of 1:4) was added for eluting the template molecule so as to obtain SMIPs1. Another 5 g of the SiO.sub.2@acrylamide was taken, and the template molecule was changed into 0.80 g of aldehyde ox-TG and 0.80 g of epoxy ox-TG separately to obtain SMIPs2 and SMIPs3 respectively with other operations same as above. FIG. 3 is a schematic diagram showing the synthesis of SMIPs. A non-imprinted polymer (SNIPs) was prepared without adding a template molecule, and other preparation steps were the same as above.

[0056] (b) Characterization of the SMIPs was conducted by Fourier infrared spectroscopy. FIG. 4 shows the infrared spectrum of SiO.sub.2 (a), amino modified SiO.sub.2 (b), SiO.sub.2@acrylamide (c), SMIPs2 (d), and SNIPs (e). According to a curve a in FIG. 4, a characteristic absorption peak of Si-O-Si appears at 1,090 cm.sup.-1, and it can be observed that a hydroxyl on the surface of an activated silica gel particle appears at 3,500 cm.sup.-1, which is conducive to the grafting of a functional monomer on the surface of the silica gel particle. According to a curve b compared with the curve a, a vibration stretching peak of —CH.sub.2— appears at 1,460 cm.sup.-1, characteristic absorption peaks of —C—H— appear at 2,930 cm.sup.-1 and 2,874 cm.sup.-1, and a characteristic absorption peak of the —NH.sub.2 group appears at 3,400 cm.sup.-1, indicating that a surface shell of SiO.sub.2 is modified by APTS. According to a curve c, a characteristic absorption peak of the —NH.sub.2 group disappears at 3,400 cm.sup.-1, and a characteristic peak of amide appears at 1,645 cm.sup.-1, indicating that the amino group is acylated successfully. From curves e and f, it can be clearly seen that the SMIPs and the SNIPs have no significant differences in the position and morphology of absorption peaks and the intensity of characteristic peaks in the spectrum diagrams, and it is proven that a template molecule on MIPs is eluted completely.

[0057] (c) The SMIPs1, the SMIPs2, and the SMIPs3 were sequentially loaded into a chromatographic column (as shown in FIG. 5) at a mass ratio of 1:1:2 (10 g: 10 g: 20 g), 2.00 g of a frying oil sample was added, and then an eluting agent (DMSO) was added for eluting all other components except for ox-TG, where the ox-TG was adsorbed onto the SMIPs.

[0058] (d) Sequential elution was conducted: eluting agents including a mixture of acetic acid and methanol at a ratio of 1:8, a mixture of acetic acid and methanol at a ratio of 1:6, and a mixture of acetic acid and methanol at a ratio of 1:4 were added in sequence for elution to obtain hydroxyl triglyceride, aldehyde triglyceride, and epoxy triglyceride.

[0059] (e) The content of the ox-TG obtained after elution was determined by a portable Raman spectrometer with potassium thiocyanate (with a characteristic peak at 2,120 cm.sup.-1) as an internal standard substance at an excitation light wavelength of 785 nm in a scanning range of 200-400 cm.sup.-1, and the test was carried out at room temperature. The epoxy ox-TG has significant characteristic peak values at 810-750 cm.sup.-1, 950-840 cm.sup.-1, and 1,280-1,240 cm.sup.-1. The hydroxyl ox-TG has wide and strong characteristic peaks at 3,700-3,200 cm.sup.-1. The aldehyde ox-TG has a significant characteristic peak value at 1,680 cm.sup.-1. The steps (c-d) were repeated for five times within one day. The ratio of the peak area of each characteristic peak to the area of an internal standard peak was calculated, the three types of ox-TG were subjected to quantitative treatment, and then the intra-day RSD of the present disclosure was obtained (Table 1).

[0060] (f) The steps (c-d) were repeatedly detected at the same time point for five consecutive days. The content of the ox-TG obtained after elution was determined by Raman spectroscopy, and the inter-day RSD based on the method was calculated (Table 1).

[0061] (g) The frying oil sample was gradually diluted to obtain various concentrations and then loaded. The steps (c-d) were repeated, and the concentration of the ox-TG in an eluent was determined by Raman spectroscopy. The lowest concentration of the ox-TG that can be detected was recorded, and the detection limit of various types of the ox-TG was obtained (with reference to Table 1).

[0062] From Table 1, it can be seen that the maximum RSD.sub.intra-day of the present disclosure is 0.6673%, the maximum RSD.sub.inter-day is 1.0270%, and the lowest detection limit is 2.0*10.sup.-6 g/ ml. The method has the advantages of high accuracy and low detection limit.

TABLE-US-00001 RSD, detection limit and recovery rate of the present disclosure Detection limit Recovery rate RSD.sub.intra-day (%) RSD.sub.inter-day (%) (g/mL) Hydroxyl ox-TG 0.6673 0.8027 1.5 × 10.sup.-6 94.3±6.5% Aldehyde ox-TG 0.8572 1.0270 2.0 × 10.sup.-6 87.5±7.2% Epoxy ox-TG 0.4125 0.3152 1.5 × 10.sup.-7 96.5±4.5%

Example 2: Selection of a Substrate Material

[0063] The silica gel as a substrate material in step (b) in Example 1 was changed into chitosan or titanium dioxide, and other conditions and parameters were consistent with those in Example 1. Results are as shown in Table 2. It can be seen that the ox-TG in the frying oil can be effectively separated when the substrate material is the silica gel, and the effect is slightly worse when other conditions are used.

TABLE-US-00002 Recovery rate of the method when different substrate materials are used Silica gel chitosan Titanium dioxide Hydroxyl ox-TG 94.3±6.5% 87.3±8.9% 92.4±9.3% Aldehyde ox-TG 87.5±7.2% 81.5±15.2% 82.9±15.0% Epoxy ox-TG 96.5±4.5% 88.5±10.5% 91.1±8.7%

Example 3: Selection of a Functional Monomer

[0064] The acrylamide as a functional monomer in step (a) in Example 1 was changed into 4-vinylpyridine (dihydroxyvinylpyridine was used as a functional monomer precursor) or methacrylic acid (when the functional monomer was methacrylic acid, the grafting of the functional monomer was required to be completed only in one step, and 3-(triethoxysilyl)propyl methacrylate was used as a raw material), and other conditions and parameters were consistent with those in Example 1. Results are as shown in Table 3. It can be seen that when the functional monomer is the methacrylic acid and the acrylamide, the present disclosure has a better effect.

TABLE-US-00003 Recovery rate of the method when different functional monomers are used Methacrylic acid Acrylamide 4-vinylpyridine Hydroxyl ox-TG 94.3±6.5% 91.4±6.7% 89.4±10.3% Aldehyde ox-TG 87.5±7.2% 89.8±9.8% 86.9±9.8% Epoxy ox-TG 96.5±4.5% 95.3±6.0% 91.8±7.1%

Example 4: Selection of an Eluting Agent

[0065] The eluting agent (dimethyl sulfoxide) in step (c) in Example 1 was changed into tetrahydrofuran or chloroform separately, and other conditions and parameters were consistent with those in Example 2. Results are as shown in Table 4. When the dimethyl sulfoxide and the tetrahydrofuran are used as the eluting agent, polar components in the frying oil can be effectively separated.

TABLE-US-00004 Recovery rate of the present disclosure when different eluting agents are used Dimethyl sulfoxide Tetrahydrofuran Chloroform Hydroxyl ox-TG 94.3±6.5% 90.2±11.2% 49.9±8.3% Aldehyde ox-TG 87.5±7.2% 109.9±14.3% 44.1±9.5% Epoxy ox-TG 96.5±4.5% 97.3±7.8% 65.3±7.8%

Example 5: Selection of the Use Amount of Frying Oil

[0066] The mass ratio of the sample (frying oil sample) to the SMIPs in step (c) in Example 1 was changed from 1:20 into 1:15, 1:25, and 1:30 separately, and other conditions and parameters were consistent with those in Example 2. Results are as shown in Table 5. When the mass ratio of the frying oil sample to the SMIPs is in the range of 1:20 to 1:25, a good separation effect is achieved.

TABLE-US-00005 Recovery rate of the present disclosure when different ratios of the sample to the SMIPs are used 1:15 1:20 1:25 1:30 Hydroxyl ox-TG 83.4±3.7% 94.3±6.5% 82.9±5.6% 79.6±4.5% Aldehyde ox-TG 83.8±3.8% 89.5±7.2% 92.9±6.2% 62.5±7.1% Epoxy ox-TG 96.4±6.0% 96.5±4.5% 90.5±10.0% 87.9±6.3%

Comparative Example 1: Comparison With a Traditional Method

[0067] FIGS. 6 (a) is a TLC image of ox-TG separated from frying oil by means of a polar substance (1), column chromatography (2) and SMIPs1 (3). FIG. 6 (b)is a hydrogen nuclear magnetic resonance spectrum image of ox-TG separated from frying oil by means of SMIPs1. According to results, it is shown that three types of ox-TG cannot be accurately separated by means of a traditional chromatographic column, while epoxy ox-TG can be effectively separated from the frying oil by means of the SMIPs1. As described in Example 1, the present disclosure has a recovery rate of 80.3-101.0%, indicating that several types of ox-TG can be accurately separated by using the method.

[0068] Although the present disclosure has been disclosed as preferred examples as above, the preferred examples are not intended to limit the present disclosure. Various changes and modifications can be made by any person familiar with the technology without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be as defined by the claims.