Sample treatment method for quantitatively detecting tocopherol content of vegetable oil

Abstract

The invention discloses a sample treatment method for quantitatively detecting tocopherol content of vegetable oil, which comprises the following steps: dissolving an oil sample to be detected in a 2mol / L potassium hydroxide-methanol solution according to a solid-to-liquid ratio of 2: 2.5, and saponifying in a water bath at 60 DEG C for 30 minutes; activating the solid-phase extraction column, and adding methanol to activate the small column; adding the saponified sample solution to a column, leaching the column with 80% methanol twice, and completely draining; receiving the eluent, adding ethyl acetate to elute a sample, drying by nitrogen, redissolving ethyl acetate, and passing through a 0.45 mu m organic filter membrane; chromatographic conditions are set, a fluorescence detector is adopted, vitamin A and vitamin E of the oil sample to be detected are determined, the excitation wavelength is 294 nm, and the emission wavelength is 328 nm. The method is good in reproducibility, simple and convenient to operate, short in detection period, small in organic reagent dosage, good in reproducibility, high in accuracy and suitable for popularization and application of the method for determining the content of the four tocopherols in the vegetable oil.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction, and in particular to a sample processing method for quantitatively detecting the tocopherol content of vegetable oil. Background Art

[0002] my country is a major producer and consumer of rapeseed, with its output ranking among the highest in the world. Rapeseed oil is the largest oil crop in my country. Rapeseed oil is rich in unsaturated fatty acids and vitamins, which are a class of organic compounds necessary for maintaining normal metabolism and function of the human body and play an important role in the metabolism of substances in the body.

[0003] In the quality control and nutritional analysis of vegetable oils, the content of tocopherol (a form of vitamin E) is a key indicator. Tocopherol not only has an antioxidant effect, but also can improve the nutritional value of vegetable oils. In addition, existing sample pretreatment techniques, such as solvent extraction, solid phase extraction, etc., often involve the use of a large amount of organic solvents, which not only burden the environment, but also may affect the health of operators, while increasing processing costs. Therefore, it is particularly important to develop a sample processing method that is simple to operate, low in cost, environmentally friendly, and can accurately determine the tocopherol content in vegetable oils. Using traditional liquid-liquid extraction, evaporation and redissolution steps, the operation steps are cumbersome, time-consuming, flammable and explosive toxic reagents are consumed, and easy emulsification leads to poor reproducibility of the method. Especially for grassroots laboratories and small oil processing enterprises, the popularization and application of these methods are greatly limited. Therefore, a sample processing method for quantitatively detecting the tocopherol content of vegetable oils is needed. Summary of the invention

[0004] The purpose of the present invention is to solve the problems of the prior art and to provide a sample processing method for quantitatively detecting the tocopherol content of vegetable oil.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention comprises the following steps:

[0007] A. Dissolve the oil sample to be tested in 2 mol / L potassium hydroxide-methanol solution at a solid-liquid ratio of 2:2.5, and saponify in a water bath at 60°C for 30 min;

[0008] For the activation of B solid phase extraction column, methanol was added to activate the column;

[0009] C. Load the saponified sample solution onto the column, and wash the column with 80% methanol for 2 times, and then completely drain it;

[0010] D receives the eluent and adds ethyl acetate to elute the sample, blows it dry with nitrogen, and passes through a 0.45 μm organic filter membrane after re-dissolving in ethyl acetate;

[0011] E. Setting the chromatographic conditions and using a fluorescence detector to measure vitamin A and vitamin E in the oil sample to be tested, wherein the excitation wavelength is 294 nm and the emission wavelength is 328 nm.

[0012] Furthermore, a solid phase extraction column using a reverse phase polymer as filler is used to purify and separate the tocopherol component in the unsaponifiable matter.

[0013] Furthermore, the chromatographic conditions are injection volume: 10 μL; chromatographic column: C30 column, column length 250 mm, inner diameter 4.6 mm, particle size 3 μm, column temperature: 20°C; mobile phase: phase A is water; phase B is methanol, elution gradient, flow rate: 0.8 mL / min.

[0014] Furthermore, for the activation of the solid phase extraction column, first add 5 mL of methanol, then add 5 mL of 80% methanol to activate the column, load the saponified sample solution onto the column, then rinse the column with 5 mL of 80% methanol for 2 times, and after completely draining, use a 10 mL centrifuge tube to receive the eluent, add 7 mL of ethyl acetate to elute the sample, blow dry with nitrogen, re-dissolve with 1 mL of ethyl acetate, filter through a 0.45 μm organic filter membrane, and wait for the machine.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention uses a reverse solid phase extraction column to replace the classic saponification-liquid-liquid extraction as a pretreatment method. The traditional liquid-liquid extraction method has the problems of complicated operation steps, long time consumption, high consumption of flammable and explosive toxic reagents, and easy emulsification resulting in poor reproducibility of the method. The method has good reproducibility, simple operation, short detection cycle, small amount of organic reagents, good method reproducibility, high accuracy, and is suitable for the promotion and use of the method for determining the content of four tocopherols in vegetable oils. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic flow chart of a sample processing method for quantitatively detecting the tocopherol content of vegetable oil proposed by the present invention;

[0018] Figure 2 This is a characteristic diagram of a solid phase extraction column with a reverse phase polymer as filler proposed by the present invention;

[0019] Figure 3 A chromatographic schematic diagram of a sample processing method for quantitatively detecting the tocopherol content of vegetable oil proposed by the present invention; DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with the embodiments and accompanying drawings. However, it should be understood that the embodiments and drawings are only used to exemplify the present invention and do not constitute any limitation on the protection scope of the present invention.

[0021] The present invention is further demonstrated below in conjunction with specific embodiments:

[0022] like Figure 1 As shown, the present invention comprises the following steps:

[0023] A. Dissolve the oil sample to be tested in 2 mol / L potassium hydroxide-methanol solution at a solid-liquid ratio of 2:2.5, and saponify in a water bath at 60°C for 30 min;

[0024] For the activation of B solid phase extraction column, methanol was added to activate the column;

[0025] C. Load the saponified sample solution onto the column, and wash the column with 80% methanol for 2 times, and then completely drain it;

[0026] D receives the eluent and adds ethyl acetate to elute the sample, blows it dry with nitrogen, and passes through a 0.45 μm organic filter membrane after re-dissolving in ethyl acetate;

[0027] E. Setting the chromatographic conditions and using a fluorescence detector to measure vitamin A and vitamin E in the oil sample to be tested, wherein the excitation wavelength is 294 nm and the emission wavelength is 328 nm.

[0028] Embodiment 1:

[0029] Oil sample to be tested: a certain brand of fish oil capsules

[0030] Solid-liquid ratio: 2:2.5 (mass of oil sample: volume of potassium hydroxide-methanol solution)

[0031] Saponification conditions: 60℃ water bath, 30min

[0032] Eluent: 80% methanol, 2 times

[0033] Eluent: Ethyl acetate

[0034] Chromatographic conditions: excitation wavelength 294nm, emission wavelength 328nm

[0035] Test results:

[0036] Vitamin A content: 250μg / g

[0037] Vitamin E content: 450μg / g

[0038] Embodiment 2:

[0039] Oil sample to be tested: a certain brand of wheat germ oil

[0040] Weigh 0.2 g (accurate to 0.0001 g) of oil sample into a centrifuge tube, add 2.5 mL of 2 mol / L potassium hydroxide-methanol solution, and saponify in a 60°C water bath for 30 min; saponification conditions: 60°C water bath, 30 min

[0041] Eluent: 80% methanol, 2 times

[0042] Eluent: Ethyl acetate

[0043] Chromatographic conditions: excitation wavelength 294nm, emission wavelength 328nm

[0044] Test results:

[0045] Vitamin A content: 120μg / g

[0046] Vitamin E content: 800μg / g

[0047] In this experiment, the use of solid phase extraction column activated the small column by methanol, then the saponified sample solution was loaded on the column, and eluted with 80% methanol, which effectively removed impurities in the sample and improved the accuracy and sensitivity of the detection. Solid phase extraction technology can selectively adsorb target compounds and reduce background interference. The chromatographic conditions are injection volume: 10μL chromatographic column: C30 column, column length 250mm, inner diameter 4.6mm, particle size 3μm, column temperature: 20℃ mobile phase: phase A is water; phase B is methanol, elution gradient, flow rate: 0.8mL / min.

[0048] like Figure 2 As shown, a solid phase extraction column with a reverse phase polymer as filler is used to simultaneously purify and separate the tocopherol components in the unsaponifiable matter. The solid phase extraction column has both hydrophilic and hydrophobic functional groups, thus having good water wettability and chemical stability, a wider pH tolerance range, and is suitable for extracting tocopherol with a strong alkaline solution, and has a balanced adsorption effect on various polar and non-polar compounds. It has a large adsorption capacity, a high sample capacity, an increased adsorption capacity for most organic matter, a higher recovery rate, and the organic matter is easily quantitatively eluted after adsorption, and the detection time is short.

[0049] like Figure 3 As shown in the figure, a fluorescence detector was used to determine vitamin A and vitamin E. The fluorescence detector has the advantages of high sensitivity and high selectivity, and can accurately determine low concentrations of vitamin A and vitamin E. In this experiment, by setting a specific excitation wavelength (294nm) and emission wavelength (328nm), accurate detection of the target compounds was achieved, improving the accuracy and reliability of the analysis.

[0050] The column was eluted with 80% methanol twice to ensure that impurities were completely removed while retaining the target compound. This optimization step improved the sample purity and the accuracy of the test results. Ethyl acetate was selected as the eluent, and the sample was dried with nitrogen and filtered after reconstitution to further ensure the purity of the sample and the accuracy of the test.

[0051] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A sample processing method for quantitatively detecting the tocopherol content of vegetable oil, characterized in that: A. Dissolve the oil sample to be tested in 2 mol / L potassium hydroxide-methanol solution at a solid-liquid ratio of 2:2.5, and saponify in a water bath at 60°C for 30 min; B. Activation of solid phase extraction column: adding methanol to activate the column; C. Load the saponified sample solution onto the column, and wash the column with 80% methanol for 2 times, and then completely drain it; D receives the eluent and adds ethyl acetate to elute the sample, blows it dry with nitrogen, and passes through a 0.45 μm organic filter membrane after re-dissolving in ethyl acetate; E sets the chromatographic conditions and uses a fluorescence detector to determine vitamin A and vitamin E in the oil sample to be detected, wherein the excitation wavelength is 294 nm and the emission wavelength is 328 nm.

2. A sample processing method for quantitatively detecting the tocopherol content of vegetable oil according to claim 1, characterized in that: A solid phase extraction column using a reverse phase polymer as filler is used to purify and separate tocopherol components in unsaponifiable matter.

3. A sample processing method for quantitatively detecting the tocopherol content of vegetable oil according to claim 1, characterized in that: The chromatographic conditions are as follows: injection volume: 10 μL; chromatographic column: C30 column, column length: 250 mm, inner diameter: 4.6 mm, particle size: 3 μm; column temperature: 20°C; mobile phase: phase A is water; phase B is methanol, elution gradient, flow rate: 0.8 mL / min.

4. A sample processing method for quantitatively detecting the tocopherol content of vegetable oil according to claim 1, characterized in that: For activation of the solid phase extraction column, first add 5 mL of methanol, then add 5 mL of 80% methanol to activate the column, load the saponified sample solution onto the column, then rinse the column with 5 mL of 80% methanol for 2 times. After completely drying, use a 10 mL centrifuge tube to receive the eluent, add 7 mL of ethyl acetate to elute the sample, blow dry with nitrogen, re-dissolve with 1 mL of ethyl acetate, filter through a 0.45 μm organic filter membrane, and wait for loading on the machine.

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