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1. Antioxidants

• The Importance of Antioxidants in Food

1. Health Benefits of Antioxidants

Intake of an appropriate amount of antioxidants helps enhance the body’s resistance to diseases, protect cardiovascular health, prevent the occurrence of certain critical illnesses, and maintain normal immune function.

2. Role of Antioxidants in Delaying Aging

Antioxidants can slow down the aging process of cells. By reducing the damage of free radicals to cells, they can delay the aging of the skin and the body to a certain extent.

3. Role of Antioxidants in Disease Prevention

Studies have shown that antioxidants help reduce the risk of various chronic diseases, such as heart disease, diabetes, and neurodegenerative diseases. They protect the body from diseases by reducing oxidative stress.

• Reasons for Adding Antioxidants to Food

1. Extending Food Shelf Life

Adding antioxidants can slow down the oxidation process of food, extend its shelf life, and reduce losses caused by spoilage.

2. Maintaining Food Color and Flavor

Antioxidants can prevent food from fading and flavor changes caused by oxidation, maintaining the sensory quality of the product.

3. Improving Food Processing Stability

During food processing, antioxidants help stabilize food components, preventing oil rancidity and deterioration of other components.

• Potential Hazards of Excessive Addition

1. Health Risks

Excessive intake of antioxidants may cause digestive system disorders, allergic reactions, and even long-term health problems.

2. Regulatory Violations

Excessive addition of antioxidants in food violates food safety standards, which may lead to product recalls or legal liabilities for enterprises.

• Judgment Basis

1. GB 2760-2014 National Food Safety Standard for the Use of Food Additives.
2. GB 2760-2024 National Food Safety Standard for the Use of Food Additives.

• Importance of Accurate Determination

1. Ensuring Food Safety

Accurate determination of antioxidant content can prevent excessive use and protect consumers’ health.

2. Avoiding Economic Losses

Accurate determination results help food enterprises reasonably control costs and avoid economic losses caused by exceeding standards.

3. Complying with Regulatory Requirements

Accurate determination of antioxidant content ensures that products meet national or international food safety standards and regulations.

1. Method Principle and Instrument Configuration

Oil samples are dissolved in n-hexane, extracted with acetonitrile, and purified by solid-phase extraction column. Determination is performed by high-performance liquid chromatography, external standard method, liquid chromatography-tandem mass spectrometry, or gas chromatography-mass spectrometry.

High-performance liquid chromatography for the determination of antioxidants involves relevant detection characteristics, as follows:

Detection characteristics:

Advantage of high sensitivity: The high sensitivity of the equipment results in a lower detection limit.

Main discussion content: This study mainly focuses on the determination of antioxidants by high-performance liquid chromatography.

III. Experimental Process, Data Analysis, and Factors Affecting Recovery Rate of Antioxidant Determination, etc., as Follows:

1. Experimental Samples and Pretreatment

– Sample Selection: Four types of edible oils, namely rapeseed oil, peanut oil, soybean oil, and blended oil, are selected as samples. Different oils have different colors, but the color does not affect the final peak appearance.

– Sample Weighing Operation: Accurately weigh 1 gram of sample into a 50-milliliter centrifuge tube, dissolve the sample with 5 milliliters of n-hexane saturated layer, and vortex for 1 minute. Single-tube or multi-tube vortex equipment can be used, and multi-tube vortex can improve efficiency.

– Extraction Steps: Extract the sample with 5 milliliters of n-hexane-saturated ether, extract three times, and combine the supernatants. The number of extractions affects the extraction effect of the target substance.

– Centrifugation Operation: Centrifuge with a 3000-rpm centrifuge. A high-speed centrifuge may emulsify the oil, and a high-speed centrifuge can also be set to low speed for use.

2. Purification and Concentration

– Solid-Phase Extraction: A C18 solid-phase extraction column with a specification of 2 grams/12 milliliters is selected. It needs to be activated and equilibrated before use. During sample loading, the flow rate is controlled to 1 drop every 5-10 seconds to ensure sufficient adsorption of the target substance. There are manual and fully automatic solid-phase extraction devices on the market.

– Elution and Collection: During elution, collect the eluate into an eggplant-shaped flask for concentration. Rotary evaporation or nitrogen blowing can be selected. Rotary evaporation is fast for large-volume samples, and nitrogen blowing is easy to control for small-volume samples.

– Notes on Rotary Evaporation: The water bath temperature and pressure need to be controlled during rotary evaporation. Excessive pressure may lead to loss of target substances, such as TBHQ, which is easily lost under high temperature and excessive pressure.

– Key Points of Nitrogen Blowing: The flow rate and temperature need to be controlled during nitrogen blowing.

3. Data Comparison and Analysis

– Instrument Configuration: A 600 ultraviolet detector, pump A, and pump B are used for gradient elution, equipped with a column oven and an automatic sampler.

– Pure Standard Chromatogram: Under normal circumstances, the pure standard chromatogram should show three sharp peaks with good resolution (TBHQ, BHA, BHT). The mobile phase and column oven status affect the peak time and peak shape.

– Matrix Interference: Different matrices have different interference peaks. For example, rapeseed oil has an interference peak at 16.5. Samples need to be purified to reduce the impact of impurities on the instrument and chromatographic column.

– Linearity and Comparison: Compare the linear graphs of purified and unpurified samples; unpurified samples have high impurity peaks and solvent peaks. Compare rotary evaporation and nitrogen blowing; there is no significant difference in values when the eluate volume is 5 milliliters.

– Recovery Rate Issue: The initial recovery rate was only 20%. Analysis showed that the effluent contained 40-60% of the target substance. The recovery rate can be increased to 90% by increasing the number of extractions, collecting the effluent, increasing the amount and times of eluent, and controlling the rotary evaporation pressure.

4. Experimental Summary and Method Setting

– Summary Idea: Low recovery rate may occur in the three pretreatment steps of extraction, purification, and concentration, and data can be analyzed and sorted out according to this logic.

– Method Setting: The editing and import of the 600 instrument method are introduced, including setting parameters such as flow rate, gradient elution table, injection depth, and detection wavelength, as well as the operation of the data processing and analysis interface.