Determination of Nitrite in Meat Products

Meat products nitrite were determined in the two kinds of forbidden national standard colorimetric method of ethylenediamine and hydrochloric acid by ion chromatography
.

This training uses the naphthalene ethylenediamine hydrochloride colorimetric method (according to the second method of GB5009.

1.
Purpose requirements

(1) Learning forbidden ethylenediamine hydrochloride colorimetric assay meat nitrite principles and operation method of the content
.

(2) Master basic operation skills such as sample preparation and extraction
.

(3) Master the use methods and operating skills of the spectrophotometer
.

2.
Principles of training

The precipitated protein sample and in addition to the fat, the conditions under weakly acidic nitrite and sulfanilic acid is diazotized and then coupled to form a magenta dye and a diamine hydrochloride naphthylethyl, nitrite content was measured external standard method
.

3.
Training supplies

(1) Analyze the balance
.

(2) Food grinder
.

(3) Spectrophotometer
.

(4) Potassium ferrocyanide solution (106g/L): Weigh 1060g of potassium ferrocyanide [K ₄ Fe(CN) ₆ ·3H ₂ O], dissolve it with water, and dilute to 1000 mL
.

(5) Zinc acetate solution (220g/L): Weigh 220.
0g zinc acetate [Zn(CH ₃ COO) ₂ ], first add 30mL glacial acetic acid to dissolve it, and dilute to 1000mL with water
.

(6) Saturated borax solution (50g/L): Weigh 5.
0g of sodium borate (Na ₂ B ₄ O ₇ ·10H ₂ O), dissolve it in 100 mL of hot water, and cool it for later use
.

(7) P-sulfanilic acid solution (4g/L): Weigh 0.
4g p-sulfanilic acid (C ₁₂ H ₁₄ NO ₃ S), dissolve it in 100 mL 20% (volume fraction) hydrochloric acid , and place it in a brown bottle Mix well and store in the dark
.

(8) Naphthalene ethylene diamine hydrochloride solution (2g/L): Weigh 0.
2 g of naphthalene ethylene diamine hydrochloride (C ₁₂ H ₁₄ N ₂ ·2HCl) and dissolve it in 100 mL of water.
After mixing, place it in a brown bottle and avoid Light save
.

(9) Sodium nitrite standard solution (200ug/mL): Weigh accurately 0.
1000g of sodium nitrite dried at 110～120℃ with constant weight , add water to dissolve and transfer to a 500mL volumetric flask, dilute with water to the mark, and mix
.

(10) Standard solution of sodium nitrite (5.
0ug/mL): Immediately before use, draw 5.
00mL of standard solution of sodium nitrite, place it in a 200mL volumetric flask, and dilute to the mark with water
.

Unless otherwise specified, the reagents used in this method are of analytical grade
.

The water is secondary water or deionized water specified in GB/T6682

4.
Safety reminder

Naphthalene ethylenediamine hydrochloride has carcinogenic effects, so pay attention to safety when using it
.

5.
Operation steps

(1) Handling of samples

①Sample pretreatment: Take an appropriate amount of meat products according to the quarter method, and use a food grinder to make a homogenate for later use
.

②Extraction: Weigh 5g (accurate to 0.
01g) to make a homogenized sample (if water is added during the preparation process, it should be converted according to the amount of water added), put it in a 50mL beaker, add 12.
5mL saturated borax solution, stir evenly, Wash the sample into a 500 mL volumetric flask with about 300 mL of water at about 70°C, heat it in a boiling water bath for 15 minutes, take it out and cool it in a cold water bath, and place it to room temperature
.

③Purification of the extract: Add 5 mL of potassium ferrocyanide solution while shaking the above extract, shake well, and then add 5 mL of zinc acetate solution to precipitate the protein
.

Add water to the mark, shake well, let it stand for 30 minutes, remove the upper fat, filter the supernatant with filter paper, discard 30 mL of the initial filtrate, and use the filtrate for later use

(2) Determination of nitrite: Pipette 40.
0 mL of the above filtrate into a 50 mL colorimetric tube with a stopper, and pipette 0.
00, 0.
20, 0.
40, 0.
60, 0.
80, 1.
00, 1.
50, 2.
00, 2.
50 mL of sodium nitrite standard use solution ( (Equivalent to 0.
0, 1.
0, 2.
0, 3.
0, 4.
0, 5.
0, 7.
5, 10.
0, 12.
5ug sodium nitrite ), respectively placed in a 50mL colorimetric tube with stopper
.

Add 2 mL of p-aminobenzene sulfonic acid solution to the standard tube and sample tube , mix well, let stand for 3 to 5 minutes, add 1 mL of naphthalene ethylene diamine hydrochloride solution, add water to the mark, mix well, let stand for 15 minutes, use 2 cm In the cuvette, adjust the zero point with a zero tube, measure the absorbance at a wavelength of 538mm, and draw a standard curve for comparison

6.
Result calculation

Where X-the content of sodium nitrite (calculated as sodium nitrite) in the sample, mg/kg

m ₁ ——Determine the quality of sodium nitrite in the sample solution, ug

m ₂ ——the mass of the sample, g

V ₁ ——Total volume of sample treatment liquid, mL

V ₂ ——Volume of sample liquid for determination, mL

It is expressed as the arithmetic mean of the two independent determination results obtained under repeatability conditions, and the result retains two significant digits
.

The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean

7.

Tips

(1) When the nitrite content is high, the excess nitrite can oxidize the azo compound, turn it into yellow, and make the red disappear.
At this time, you can add the reagent first, and then drop the test solution to avoid nitrous acid.
Excessive salt
.

(2) Fat should be removed for samples with high fat content
.

The fat can be coagulated by heating and cooling and then filtered and removed, or the upper layer of fat can be extracted separately by skimming; for colored samples, such as braised meat, the colorimetric determination is affected by the pigment, and the protein should be precipitated by zinc sulfate.

(3) When the sample is precipitating protein, the zinc acetate solution should not be used too much, otherwise a white precipitate will form and affect the determination

(4) Do not leave the filtrate for too long, so as to avoid the oxidation of nitrite that will affect the measurement results

Related link: Determination of color retention agent