Complete screening method of commonly used preservatives (antioxidants, preservatives) in food

Commonly used food preservatives include benzoic acid, sorbic acid, parabens, calcium propionate (sodium), calcium lactate (sodium), sodium diacetate, dehydroacetic acid, etc.

Preservatives that have antiseptic effects but are not currently used as food additives and are easily abused include salicylic acid, p-hydroxybenzoic acid and m-hydroxybenzoic acid.

Freshness and antioxidants include butylhydroxyanisole (BHA), 2,6-di-tert-butyl-p-cresol (BHT) and tert-butyl hydroquinone (TBHQ), p-tert-butylphenol, 2-benzene Phenol, 2-naphthol, 4-phenylphenol, etc.

Food preservatives play a preservative role mainly in three ways: 1. inhibit the formation of microbial cell walls; 2. affect the function of cell membranes; 3. inhibit protein synthesis or denature proteins.

Each preservative mainly acts on microorganisms in one way. Therefore, the "antibacterial spectrum" of a single preservative will be relatively small, and it will often require a relatively large concentration, which is easily exceeded.

Many foods, especially nutrient-rich foods, often use a variety of preservatives in order to expand the "antibacterial spectrum", reduce the concentration of a single preservative, improve the antiseptic effect, and extend the shelf life of the food. For example, benzoic acid and trisorbic acid preservatives are often used in combination to improve the antiseptic effect.

Some of the current national standard methods detect multiple preservatives at the same time, such as "National Food Safety Standard GB 21073-2010 Determination of Benzoic Acid and Sorbic Acid in Milk and Dairy Products", etc. It is very reasonable to formulate corresponding measures based on the characteristics of the food industry Detection method.

However, there are some standards that only detect a certain preservative, and there is no corresponding detection regulation for some "legal" preservatives that are beyond the scope of use, which is likely to cause "unsafe products that meet the standards"; and For some illegal preservatives, there is currently no corresponding detection method.

In response to this phenomenon, combined with the physicochemical properties of the preservatives, we have developed a set of solutions that strives to analyze as many preservatives as possible in a single step.

First of all, we divide preservatives into two categories: water-soluble and fat-soluble. Water-soluble preservatives mainly include benzoic acid, hydroxybenzoic acid (illegal preservatives), sorbic acid, small molecule organic acids, etc. (lactic acid, propionic acid, diacetic acid), as long as these preservatives are used for water-soluble or Water-soluble foods, such as drinks, cakes, seasonings, etc.

Fat-soluble preservatives (preservatives) mainly include parabens, butylated hydroxyanisole (BHA), 2,6-di-tert-butyl p-cresol (BHT) and tert-butyl hydroquinone (TBHQ), For p-tert-butylphenol, 2-phenylphenol, 2-naphthol, 4-phenylphenol, etc., these additives are mainly used in foods containing more oil or dried fruits.

Preservatives (preservatives) can only exert antiseptic and fresh-keeping effects when they are fully dissolved or evenly distributed in the food medium. Therefore, according to the condition of the food matrix, it can be roughly determined which type of preservatives (preservatives) may be included, and the appropriate extraction is selected. Purification method, measured.

The characteristics of this scheme are: no matter whether the preservative (preservative) is water-soluble or fat-soluble, the same purification small column and the same HPLC chromatography column are used, which has a very wide applicability and is convenient.

1 Experimental part

1.1 Instruments and reagents

HPLC chromatograph 1 (Agilent 1200)

HPLC chromatograph 2 (HITACHI L2000)

SPE decompression device (Phenomenex)

SPE purification cartridge: Strata-XA 200mg / 3mL (P / N 8B-S123-FBJ)

For large samples or samples rich in organic acids, choose Strata-XA 500mg / 3mL (P / N 8B-S123-HBJ)

HPLC column: Synergi Polar-RP 4μ 250 × 4.6mm (P / N 00G-4336-E0)

Water-soluble preservative solution:

extract:

1. Aqueous solution sample: take 10mL of sample accurately, add 0.1g of sodium bicarbonate, fully dissolve, ultrasonically remove the sample containing CO2, use pH test paper to detect the pH of the solution is about 7 (if pH <7, continue adding sodium carbonate to adjust the pH to 7) , 0.45μm filter membrane is filtered, and the filtrate is used as the solution to be purified.

2. Semi-solid samples and solid samples: take 1 to 5g of the sample precisely, add 10 to 20mL of 0.1M sodium bicarbonate solution, dissolve it by ultrasound, and filter the solution with a 0.45μm filter membrane, and use the filtrate as the solution to be purified.

Purification:

SPE cartridge: Strata-XA 200mg / 3mL (P / N 8B-S123-FBJ)

Activation balance: 3mL methanol, 3mL 0.1M sodium bicarbonate (pH 7)

Sample loading: 2 ～ 5mL liquid to be purified

Cleaning 1: 3mL of water

Cleaning 2: 3mL of methanol

Blow dry 1min

Elution: 2mL × 2 0.1% HCL methanol

Combine the eluent into a 5mL volumetric flask, add water to the mark, shake well, filter with 0.45um filter membrane, and continue the filtrate as the test solution.

HPLC determination:

Instruments: Agilent 1200 quaternary low-pressure gradient pump, UV-visible detector

Column: Synergi Polar-RP 4μ 250 × 4.6mm (P / N 00G-4336-E0)

Protection column: KJ0-4282 AJ0-6076

Detection wavelength: 0 ～ 9min, 200nm; 9.1 ～ 11min, 270nm; 11.1 ～ 25min, 230nm.

Injection volume: 5μL

Standard product concentration: lactic acid, acetic acid, propionic acid 1.0mg / ml, p-hydroxybenzoic acid, 3-hydroxybenzoic acid, sorbic acid, benzoic acid, salicylic acid, fumaric acid, gallic acid 0.1mg / ml, the solvent is water . (The substance in red is an illegal additive).

Mobile phase and gradient program:

Time 20mM potassium dihydrogen phosphate (:

N 8B-S123-FBJ)

Activation balance: 3mL methanol, 3mL sodium hydroxide solution

Sample loading: 2 ～ 5mL liquid to be purified

Cleaning 1: 0.1M sodium hydroxide solution 3mL

Cleaning 2: 0.1M sodium hydroxide solution methanol solution 3mL

Blow dry 1min

Elution: 2mL × 2 0.1% HCl methanol

Combine the eluent into a 2mL volumetric flask, add water to the mark, shake well, filter with 0.45um filter membrane, and continue the filtrate as the test solution.

HPLC determination:

Instruments: HITACHI L2000 quaternary gradient pump, L2300 UV-visible detector

Column: Synergi Polar-RP 4μ 250 × 4.6mm (P / N 00G-4336-E0)

Protection column: KJ0-4282 AJ0-6076

Detection wavelength: 280nm

Injection volume: 10 μL

Standard concentration: 0.1mg / mL solvent is methanol

Mobile phase and gradient program:

12 standard maps of fat-soluble preservatives

Standard recovery rate:

10 water-soluble preservatives

12 fat-soluble preservatives and antioxidants

name

Recovery rate

RSD n = 5

name

Recovery rate

RSD n = 5

Lactic acid

83.3%

4.3%

Propyl gallate

97.3.2%

2.4%

Acetic acid

76.7%

5.2%

Methylparaben

93.3%

2.7%

Fumaric acid

93.0%

2.1%

Ethyl p-hydroxybenzoate

92.8%

2.7%

Propionic acid

78.4%

4.7%

Tert-butyl hydroquinone (TBHQ)

94.2%

2.1%

Gallic acid

95.2%

2.2%

Propylparaben

95.9%

2.3%

P-hydroxybenzoic acid

94.9%

1.9%

2-naphthol

96.7%

2.6%

M-hydroxyphenyl acid

97.1%

2.1%

Butyl p-hydroxyphenylhexanoate

94.6%

2.0%

Sorbic acid

90.8%

2.7%

4-tert-butylphenol

93.5%

3.1%

benzoic acid

98.3%

1.8%

4-phenylphenol

96.6%

3.7%

Salicylic acid

98.2%

1.7%

2-phenylphenol

92.4%

3.4%

Butyl Hydroxyanisole (BHA)

97.5%

2.5%

2,6-di-tert-butyl-p-cresol (BHT)

96.9%

2.3%

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