Method for determining nine antioxidants in food by using SPE-HPLC (Solid Phase Extraction-High Performance Liquid Chromatography) method

Abstract

The invention discloses a method for determining nine antioxidants in food by using an SPE-HPLC (Solid Phase Extraction-High Performance Liquid Chromatography) method, which comprises the following steps: a, extracting a sample: i, dissolving the sample: dissolving 1.0 g of the sample by using acetonitrile-saturated normal hexane, carrying out vortex mixing for 1 minute, and standing for 10 minutes; ii, extracting: adding 20mL of acetonitrile, carrying out vortex oscillation extraction for 10 minutes, cooling at low temperature for 5 minutes, centrifuging at 6000 rpm for 5 minutes, and taking out a lower acetonitrile layer; and iii, repeated extraction: adding 20mL of acetonitrile into the residue, repeatedly extracting twice, combining the three extracting solutions, and remarkably improving the detection sensitivity and accuracy by optimizing the extraction and purification steps. The method is not only suitable for antioxidant determination of various oil-containing foods, but also effectively solves the problems of long sample treatment time, low extraction efficiency, unstable detection and the like in the prior art.

Description

Technical Field

[0001] The invention relates to the field of food detection, and in particular to a method for determining 9 antioxidants in food by using a SPE-HPLC method. Background Art

[0002] Edible oil plays an important role in human health, providing the body with essential fatty acids and promoting the absorption of fat-soluble vitamins. However, the oxidation and rancidity of oils and fats is an important cause of edible oil deterioration, which will lead to changes in the sensory properties of oils and fats, adverse effects on human health, and may even be carcinogenic. In order to prevent the oxidation of oils and fats, antioxidants are usually added to food. However, long-term excessive use of antioxidants can cause harm to human health. For example, propyl gallate (PG) can cause kidney damage or contact dermatitis, tert-butylhydroquinone (TBHQ) may cause cancer, and tert-butyl-p-hydroxyanisole (BHA) and 2,6-di-tert-butyl-p-methylphenol (BHT) are also included in the list of carcinogens. Therefore, strict monitoring and testing of antioxidants in edible oils and oil-containing products is very necessary. Summary of the invention

[0003] The purpose of the present invention is to provide a method for determining 9 antioxidants in food using the SPE-HPLC method, which has the advantage of being applicable to the determination of antioxidants in a variety of oil-containing foods and solves the problems raised in the background technology.

[0004] To achieve the above object, the present invention provides the following technical scheme: a method for determining 9 antioxidants in food by SPE-HPLC, comprising the following steps: a. sample extraction: i. dissolving sample: dissolving 1.0 g sample in n-hexane saturated with acetonitrile, vortex mixing for 1 minute and then standing for 10 minutes; ii. extraction: adding 20 mL of acetonitrile and vortexing for extraction for 10 minutes, low temperature cooling for 5 minutes, centrifuging at 6000 rpm for 5 minutes, and taking out the lower acetonitrile layer; iii. repeated extraction: adding 20 mL of acetonitrile to the residue, repeating the extraction twice, and combining the three extracts;

[0005] b. Sample purification: i. Add to purification tube: Add the combined extract to a 50 mL purification tube, vortex mix for 1 minute, and centrifuge at 8000 rpm for 5 minutes; ii. Concentration: Take 15 mL of supernatant in a graduated glass centrifuge tube, blow nitrogen gas to less than 1 mL in a 40°C water bath, make up to 1 mL with acetonitrile, and vortex mix; iii. Freeze filtration: Freeze at -4°C for 30 minutes, take the supernatant and filter it through a 0.45 μm PTFE filter membrane;

[0006] c. Analysis and detection: i. Use chromatographic column: flow rate is 1.0mL / min, injection volume is 5μL, column temperature is 35℃; ii. Mobile phase: methanol and 0.5% formic acid aqueous solution; iii. Gradient setting: 0-5 minutes A: 50%, B: 50%; 5-10 minutes A: 70%, B: 30%; 10-20 minutes A: 92%, B: 8%; 20-25 minutes A: 100%, B: 0%; 25-35 minutes A: 50%, B: 50%.

[0007] Preferably, the 9 antioxidants include: propyl gallate (PG), 2,4,5-trihydroxybutyrophenone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), tert-butylparahydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butylparamethylphenol (BHT), and dodecyl gallate (DG).

[0008] Preferably, 2 g of anhydrous sodium sulfate is added to the ham and canned peanut samples before extraction to disperse them, and then 2.5 mL of n-hexane (saturated with acetonitrile) is added.

[0009] Preferably, the solid sample needs to be dispersed by adding anhydrous sodium sulfate, and needs to be refrigerated for 30 minutes during the extraction, purification and volume determination process to prevent oil precipitation.

[0010] Preferably, in the recovery rate experiment, the vortex mixing intensity during sample extraction should be above 2000 rpm, and the supernatant after purification should be concentrated to less than 1 mL.

[0011] Preferably, the sample recovery rate ranges from 80% to 110%.

[0012] Preferably, it is suitable for the determination of propyl gallate (PG), 2,4,5-trihydroxybutyrophenyl (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT) and dodecyl gallate (DG) in foods such as peanut oil, sunflower oil, olive oil, blended oil, sesame oil, palm oil, lard, fried nuts, fried dough twist, instant noodles, potato chips, ham, oatmeal, canned peanuts, etc.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for determining 9 antioxidants in food by SPE-HPLC, which significantly improves the sensitivity and accuracy of detection by optimizing the extraction and purification steps. The method is not only applicable to the determination of antioxidants in a variety of oil-containing foods, but also effectively solves the problems of long sample processing time, low extraction efficiency and unstable detection in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the structure of the present invention; DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] A method for determining 9 antioxidants in food by SPE-HPLC, comprising the following steps: a. sample extraction: i. dissolving the sample: dissolving 1.0 g of the sample in n-hexane saturated with acetonitrile, vortexing for 1 minute and then standing for 10 minutes; ii. extracting: adding 20 mL of acetonitrile, vortexing and extracting for 10 minutes, low-temperature cooling for 5 minutes, centrifuging at 6000 rpm for 5 minutes, and taking out the lower acetonitrile layer; iii. repeating the extraction: adding 20 mL of acetonitrile to the residue, repeating the extraction twice, and combining the three extracts;

[0017] b. Sample purification: i. Add to purification tube: Add the combined extract to a 50 mL purification tube, vortex mix for 1 minute, and centrifuge at 8000 rpm for 5 minutes; ii. Concentration: Take 15 mL of supernatant in a graduated glass centrifuge tube, blow nitrogen gas to less than 1 mL in a 40°C water bath, make up to 1 mL with acetonitrile, and vortex mix; iii. Freeze filtration: Freeze at -4°C for 30 minutes, take the supernatant and filter it through a 0.45 μm PTFE filter membrane;

[0018] c. Analysis and detection: i. Use chromatographic column: flow rate is 1.0mL / min, injection volume is 5μL, column temperature is 35℃; ii. Mobile phase: methanol and 0.5% formic acid aqueous solution; iii. Gradient setting: 0-5 minutes A: 50%, B: 50%; 5-10 minutes A: 70%, B: 30%; 10-20 minutes A: 92%, B: 8%; 20-25 minutes A: 100%, B: 0%; 25-35 minutes A: 50%, B: 50%.

[0019] Time (min) 0 5 5.1 10 10.1 20 20.1 25 25.1 35 A(％) 50 50 70 70 92 75 100 100 50 50 B(％) 50 50 30 30 8 25 0 0 50 50

[0020] Preferably, the 9 antioxidants include: propyl gallate (PG), 2,4,5-trihydroxybutyrophenone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT), and dodecyl gallate (DG).

[0021] Preferably, 2 g of anhydrous sodium sulfate is added to the ham and canned peanut samples before extraction to disperse them, and then 2.5 mL of n-hexane (saturated with acetonitrile) is added.

[0022] Preferably, the solid sample needs to be dispersed by adding anhydrous sodium sulfate, and needs to be refrigerated for 30 minutes during the extraction, purification and volume determination process to prevent oil precipitation.

[0023] Preferably, in the recovery rate experiment, the vortex mixing intensity during sample extraction should be above 2000 rpm, and the supernatant after purification should be concentrated to less than 1 mL.

[0024] Preferably, the sample recovery rate ranges from 80% to 110%.

[0025] Preferably, it is suitable for the determination of propyl gallate (PG), 2,4,5-trihydroxybutyrophenyl (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT) and dodecyl gallate (DG) in foods such as peanut oil, sunflower oil, olive oil, blended oil, sesame oil, palm oil, lard, fried nuts, fried dough twist, instant noodles, potato chips, ham, oatmeal, canned peanuts, etc.

[0026] Among them, the added concentration was 400 mg / kg (spiked amount: 1000 ug / mL, 0.4 mL);

[0027] Addition concentration 200 mg / kg (spiking amount: 1000 ug / mL, 0.2 mL);

[0028] The added concentration was 20 mg / kg (spiking amount: 200 ug / mL, 0.1 mL).

[0029] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining 9 antioxidants in food using SPE-HPLC, characterized in that: The following steps are involved: a. Sample extraction: i. Dissolve the sample: Use acetonitrile-saturated n-hexane to dissolve 1.0 g of sample, vortex mix for 1 minute and let stand for 10 minutes; ii. Extract: Add 20 mL of acetonitrile and vortex for 10 minutes, cool for 5 minutes, centrifuge at 6000 rpm for 5 minutes, and remove the lower acetonitrile layer; iii. Repeat the extraction: Add 20 mL of acetonitrile to the residue, repeat the extraction twice, and combine the three extracts; b. Sample purification: i. Add to purification tube: Add the combined extract to a 50 mL purification tube, vortex mix for 1 minute, and centrifuge at 8000 rpm for 5 minutes; ii. Concentration: Take 15 mL of supernatant in a graduated glass centrifuge tube, blow nitrogen gas to less than 1 mL in a 40°C water bath, make up to 1 mL with acetonitrile, and vortex mix; iii. Freeze filtration: Freeze at -4°C for 30 minutes, take the supernatant and filter it through a 0.45 μm PTFE filter membrane; c. Analysis and detection: i. Use chromatographic column: flow rate is 1.0mL / min, injection volume is 5μL, column temperature is 35℃; ii. Mobile phase: methanol and 0.5% formic acid aqueous solution; iii. Gradient setting: 0-5 minutes A: 50%, B: 50%; 5-10 minutes A: 70%, B: 30%; 10-20 minutes A: 92%, B: 8%; 20-25 minutes A: 100%, B: 0%; 25-35 minutes A: 50%, B: 50%.

2. The method for determining 9 antioxidants in food by SPE-HPLC according to claim 1, characterized in that: The nine antioxidants include propyl gallate (PG), 2,4,5-trihydroxybutyrophenone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT), and dodecyl gallate (DG).

3. The method for determining 9 antioxidants in food by SPE-HPLC according to claim 1, characterized in that: Before extraction, 2 g of anhydrous sodium sulfate was added to the ham and canned peanut samples to disperse them, and then 2.5 mL of n-hexane (saturated with acetonitrile) was added.

4. The method for determining 9 antioxidants in food by SPE-HPLC according to claim 1, characterized in that: Solid samples need to be dispersed by adding anhydrous sodium sulfate, and need to be refrigerated for 30 minutes during the extraction, purification and volume determination process to prevent oil precipitation.

5. The method for determining 9 antioxidants in food by SPE-HPLC according to claim 1, characterized in that: In the recovery rate experiment, the vortex mixing intensity should be above 2000rpm during sample extraction, and the supernatant after purification should be concentrated to less than 1mL.

6. The method for determining 9 antioxidants in food by SPE-HPLC according to claim 1, characterized in that: Sample recoveries ranged from 80% to 110%.

7. The method for determining 9 antioxidants in food by SPE-HPLC according to claim 1, characterized in that: It is suitable for the determination of propyl gallate (PG), 2,4,5-trihydroxybutyrophenyl (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT) and dodecyl gallate (DG) in foods such as peanut oil, sunflower oil, olive oil, blended oil, sesame oil, palm oil, lard, fried nuts, fried dough twist, instant noodles, potato chips, ham, oatmeal, canned peanuts, etc.

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