Food Nitrite Content Assay Kit (NED, Micro Method)

Cat. No.: N1507975

GRADE & PURITY BioReagent

Overview

In food products, nitrite can bind with myoglobin in meat to form a more stable compound. In the food processing industry, it is used as a color‑fixing agent to maintain the appealing appearance of meat products, prevent the growth of Clostridium botulinum, and enhance the safety of edible meat products. However, long‑term consumption of foods containing excessive nitrite may induce digestive system cancers.

Detection Principle

Under acidic conditions, nitrite reacts with sulfanilic acid to form a diazo compound, which then couples with N‑(1‑naphthyl)ethylenediamine to produce a purple‑red azo dye with a characteristic absorption peak at 540 nm.

Detection Range: 1.563‑100 µM

Sensitivity: 1.563 µM

 Note: Before formal testing, it is recommended to perform a preliminary experiment with 2‑3 samples expected to show significant differences.

Reagents, consumables and Equipments not provided

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)

• 96‑well plate or micro‑volume glass cuvette, adjustable pipettes and tips

• Ice maker, centrifuge, water bath

• Deionized water

• Mortar

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Extraction Solution Ⅰ	Ready‑to‑use	Store at room temperature. This is a saturated solution; slight precipitation is normal. Shake well before use.
Extraction Solution Ⅱ	Ready‑to‑use	Store at room temperature.
Extraction Solution Ⅲ	Ready‑to‑use	Store at room temperature.
Activated Carbon	Ready‑to‑use	Store at room temperature.
Reagent Ⅰ	Ready‑to‑use; equilibrate to room temperature before use	Store at 4 °C protected from light.
Reagent Ⅱ	Ready‑to‑use; equilibrate to room temperature before use	Store at 4 °C protected from light.
NaNO₂ Standard (1M)		Store at 4 °C.

Note: Reagent Ⅱ and NaNO₂ Standard (1 M) are somewhat toxic; perform experiments in a fume hood.

2. Standard Curve Setup

Dilute the 1 M NaNO₂ Standard to 100 µM using deionized water. Then, dilute the 100 µM NaNO₂ Standard as shown in the table below to obtain 50, 25, 12.5, 6.25, 3.125, and 1.563 µM standards.

Tube	Standard Volume	Deionized Water Volume (µL)	Standard Concentration (µM)
Std.1	200µL of 100μM NaNO₂ Standard	0	100
Std.2	100µL of Std.1 (100μM)	100	50
Std.3	100µL of Std.2 (50μM)	100	25
Std.4	100µL of Std.3 (25μM)	100	12.5
Std.5	100µL of Std.4 (12.5μM)	100	6.25
Std.6	100µL of Std.5 (6.25μM)	100	3.125
Std.7	100µL of Std.6 (3.125μM)	100	1.563

Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3. Sample Preparation

Weigh approximately 0.2 g fresh weight or 0.05 g dry weight of sample, homogenize, add 0.5 mL of Extraction Solution Ⅰ, incubate in a boiling water bath for 15 minutes, cool to room temperature, add 0.5 mL of Extraction Solution Ⅱ, vortex to mix, add 0.5 mL of Extraction Solution Ⅲ, add a small amount of Activated Carbon (about 1 mg) using forceps, mix well, let stand for 30 minutes, centrifuge at 8,000 g, 25 °C for 15 minutes, and collect the supernatant for testing.

Note: After adding Extraction Solutions Ⅱ and Ⅲ, the sample may become viscous, which is normal.

4. Experimental Steps

4.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 540 nm. Zero the visible spectrophotometer with deionized water.

4.2 Operation table (perform the following steps in a 96‑well plate or micro‑volume glass cuvette):

Reagent (µL)	Test Well	Standard Well	Blank Well
Sample	70	0	0
Standard	0	70	0
Deionized water	0	0	70
Reagent Ⅰ	65	65	65
Reagent Ⅱ	65	65	65

Mix well, incubate at 25 °C for 15 minutes, measure the absorbance at 540 nm, recorded as A_Test, A_Standard, and A_Blank. Calculate ΔA_Test = A_Test – A_Blank and ΔA_Standard = A_Standard – A_Blank.

Note: The blank and standard wells need to be measured only once. It is recommended to perform a preliminary experiment with 2‑3 samples showing expected large differences before formal testing. If ΔA_Test is less than 0.001, appropriately increase the sample amount. If ΔA_Test is greater than 1.0, further dilute the sample with deionized water and multiply the result by the dilution factor, or reduce the amount of sample used for extraction.

5. Calculation of Results

Note: We provide two formula sets for your convenience: the derived calculation formulas and the simplified formulas. They are completely equivalent. The simplified formulas are recommended as the final calculation formulas.

5.1 Plotting the Standard Curve

Plot the standard curve with the standard concentration as the y‑axis and ΔA_Standard as the x‑axis. Substitute ΔA_Test into the standard curve equation to obtain y (µM).

5.2 Calculation of Nitrite Content in Samples

NO₂⁻ (µg/g) = y × V_Total × 46 ÷ 1,000 ÷ W × n = 0.069 × y ÷ W × n

Parameter Description:

V_Total: Total volume of the sample extract, 1.5 mL;

46: Molecular weight of NO₂⁻, g/mol;

1,000: Unit conversion factor, 1 L = 1,000 mL;

W: Sample mass, g;

n: Dilution factor of the sample.

6. Example Results

Typical Standard Curve:

Experimental Examples:

For 0.2 g of pickled vegetable measured using a 96‑well plate: ΔA = 0.103. Substituting into the equation yields y = 10.345 µM. The calculated nitrite content is: NO₂⁻ (µg/g) = 0.069 × 10.345 ÷ 0.2 × 1 = 3.569 µg/g.

For 0.2 g of canned meat measured using a 96‑well plate: ΔA = 0.007. Substituting into the equation yields y = 0.75 µM. The calculated nitrite content is: NO₂⁻ (µg/g) = 0.069 × 0.75 ÷ 0.2 × 1 = 0.259 µg/g.

Notes

This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. N1507975A, N1507975B, N1507975C, N1507975D: Store at room temperature long term (12 months). N1507975E, N1507975F: Store at 2-8℃ long term (12 months). Store in the dark. N1

Contents & Storage

N1507975	Component	96T	Storage
N1507975A	Extraction Solution Ⅰ	70 mL	RT.
N1507975B	Extraction Solution Ⅱ	70 mL	RT.
N1507975C	Extraction Solution Ⅲ	70 mL	RT.
N1507975D	Activated Carbon	120 mg	RT.
N1507975E	Reagent Ⅰ	10 mL	2-8℃. Store in the dark.
N1507975F	Reagent Ⅱ	10 mL	2-8℃. Store in the dark.
N1507975G	NaNO₂ Standard (1M)	500 μL	2-8℃