🍎 Nitrates and nitrites in meat products — reduction with nitrate reductase, the Griess reaction, spectrometry at 540 nm
In short
An aqueous extract of 10 g of the homogenised sample (pH 8. 0–8. The test is performed according to PN-EN 12014-3:2006; Range of the validation: 9–22 mg/kg of nitrites (NaNO2) and 23–48 mg/kg of nitrates (NaNO3); applicability 5–125 mg/kg of the sum as NaNO2. The procedure comprises 9 steps (about 46–46 min in total); it is used for: Control of the content of nitrites and nitrates (E249–E252) in cured meats, canned goods and delicatessen products, Official supervision of the limits of additives in meat products, Studies of curing and of the transformations of nitrates during the ripening of cured meats.
At a glance
- Standard: PN-EN 12014-3:2006
- Category: Food chemistry
- Procedure steps: 9
- Total time of stages: 46 min–46 min
- Range of the validation: 9–22 mg/kg of nitrites (NaNO2) and 23–48 mg/kg of nitrates (NaNO3); applicability 5–125 mg/kg of the sum as NaNO2
- Test portion and extraction: 10 g (to 10 mg), 50 ml of water + 50 ml of hot water, pH 8.0–8.5, 15 min in a boiling bath, a 200 ml flask
- Clarification: 4 ml each of Carrez solutions no. 1 and no. 2; a fluted filter paper, the first 20 ml of the filtrate to be discarded
Overview
The standard specifies a spectrometric method of determining the content of nitrates and nitrites in meat products, validated for various products with a content of 9–22 mg/kg of nitrites (as sodium nitrite) and 23–48 mg/kg of nitrates (as sodium nitrate); experience shows that the method is also suitable for a total content of nitrites and nitrates from 5 to 125 mg/kg calculated as sodium nitrite. EN 12014-3:2005 replaced ENV 12014-3:1998 and belongs to the series „Determination of nitrate and/or nitrite content" (part 2: HPLC/IC for vegetables, part 4: IC for meat products, part 5: the enzymatic method for infant food, part 7: the flow method after cadmium reduction). The description is based on the preview of EN 12014-3:2005 (up to point 6.2.1); PN-EN 12014-3:2006 with the corrigendum Ap1:2008 is in the list of testing procedures of the QAFP branch booklet for canned meat.
Sodium and potassium nitrate(V) (E251, E252) and potassium and sodium nitrite (E249, E250) are used as additives in meat processing: the nitrites give the products the pink curing colour (nitrosylmyoglobin), aroma and curing flavour, act as antioxidants and inhibit the development of Clostridium botulinum and C. perfringens, while the nitrates are their reservoir, from which the nitrites arise during curing; sodium nitrite is however potentially carcinogenic (nitrosamines) and causes methaemoglobinaemia. According to the regulations cited in Bromatologia i Chemia Toksykologiczna (2009), nitrites are used only in a mixture with NaCl containing 0.5–0.6 % NaNO2, in salami the content of nitrites is to be below 60 mg/kg, and the sum of nitrites and nitrates must not exceed 600 mg/kg; in the other cured meats, delicatessen products and pasteurised canned goods the total content must not exceed 125 mg/kg.
The principle of the determination of nitrites is based on the Griess reaction: the nitrites form with sulfanilamide a diazonium salt, which couples with N-(1-naphthyl)ethylenediamine into a red azo dye; the nitrates in the extract are reduced to nitrites with nitrate reductase (instead of a cadmium column) and calculated from the difference of two measurements.
Method principle
A homogenised sample (a temperature below 25 °C; a mincer with holes ≤ 4.5 mm, passed through twice) in an amount of 10 g (to 10 mg) is homogenised with about 50 ml of water for 30–60 s, rinsed with 50 ml of hot water, the pH is brought to 8.0–8.5 with a solution of NaOH of about 1 mol/l and it is heated for 15 min in a boiling bath, with shaking. After cooling the extract is transferred into a 200 ml flask, 4 ml each of Carrez solutions no. 1 (150 g of potassium hexacyanoferrate(II) in 1 l) and no. 2 (230 g of zinc acetate in 1 l) are added, it is made up with water and filtered through a fluted filter paper free of nitrates and nitrites, the first 20 ml being discarded. Nitrites: 2.0 ml of the solution is mixed with 1.0 ml of water and 3.0 ml of the mixture of colour reagents (equal volumes of a solution of 8 g of sulfanilamide in 1 l with 330 ml of HCl and of a solution of 0.330 g of N-(1-naphthyl)ethylenediamine dihydrochloride in 250 ml), kept in the dark and after 30 min the absorbance is measured at 540 nm against water. Nitrates: a portion of the extract is incubated with an imidazole buffer of pH 7.8, NADPH with FAD and nitrate reductase (Aspergillus sp., EC 1.6.6.2) — the nitrites formed together with the original ones react with the colour reagents; the content of nitrates is calculated from the difference. The calibration curves are prepared from solutions of sodium nitrite (0.13–0.53 mg NO2− in 200 ml) and potassium nitrate (0.14–0.55 mg calculated as nitrite), prepared with Carrez clarification like the samples.
Applications
- Control of the content of nitrites and nitrates (E249–E252) in cured meats, canned goods and delicatessen products
- Official supervision of the limits of additives in meat products
- Studies of curing and of the transformations of nitrates during the ripening of cured meats
- A testing procedure of the QAFP quality system for canned meat
- A cadmium-free method — an alternative to cadmium reduction in laboratories limiting toxic waste
Key parameters
| Parameter | Value |
|---|---|
| Range of the validation | 9–22 mg/kg of nitrites (NaNO2) and 23–48 mg/kg of nitrates (NaNO3); applicability 5–125 mg/kg of the sum as NaNO2 |
| Test portion and extraction | 10 g (to 10 mg), 50 ml of water + 50 ml of hot water, pH 8.0–8.5, 15 min in a boiling bath, a 200 ml flask |
| Clarification | 4 ml each of Carrez solutions no. 1 and no. 2; a fluted filter paper, the first 20 ml of the filtrate to be discarded |
| Colour reaction | 2.0 ml of the solution + 1.0 ml of water + 3.0 ml of the mixture of reagents, 30 min in the dark, 540 nm against water |
| Enzymatic reduction | An imidazole buffer of pH 7.8; 125 mg of NADPH-Na4 and 2.5 mg of FAD in 50 ml of buffer; 11 mg of nitrate reductase (about 0.4 U/mg) in 10 ml of buffer with 50 mg of EDTA (stable for about 14 days at 4 °C) |
| Standards | NaNO2 400 mg/l → 20 mg/l → 0.13; 0.27; 0.40; 0.53 mg NO2−/200 ml; KNO3 600 mg/l → 30 mg/l → 0.14–0.55 mg as nitrite |
Standard
- Standard number
- PN-EN 12014-3:2006
- Title (PL)
- Artykuły żywnościowe — Oznaczanie zawartości azotanów i/lub azotynów — Część 3: Spektrometryczne oznaczanie zawartości azotanów i azotynów w produktach mięsnych po enzymatycznej redukcji azotanów do azotynów
- Title (EN)
- Foodstuffs — Determination of nitrate and/or nitrite content — Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite
Step-by-step procedure
-
Homogenisation
Homogenise the laboratory sample so that the temperature does not exceed 25 °C; with a mincer pass it through at least twice.
-
Extraction
Weigh out 10 g (to 10 mg) into a wide-necked conical flask, add about 50 ml of water, homogenise for 30–60 s, rinse the shaft of the homogeniser with 50 ml of hot water. Set the pH to 8.0–8.5 with a solution of NaOH (a pH meter); for raw products do not exceed 8.5, for scalded and cooked ones one may go up to about 9.5.
-
Heating
Heat the flask for 15 min in a boiling water bath, shaking it several times; cool it to room temperature.
-
Clarification
Transfer quantitatively into a 200 ml volumetric flask, add 4 ml each of Carrez solution no. 1 and no. 2, shaking after each addition; make up with water, mix, filter through a fluted filter paper, discarding the first 20 ml of the filtrate (the sample solution).
-
Standard solutions
Into a series of 200 ml flasks measure out 10, 20, 30 and 40 ml of the diluted solution of sodium nitrite (0.13; 0.27; 0.40; 0.53 mg NO2−) and — separately — of potassium nitrate (0.14; 0.27; 0.41; 0.55 mg as nitrite), set the pH to 8.0–8.5, add 4 ml each of Carrez no. 1 and no. 2, make up, filter, discarding the first 20 ml.
-
Determination of the nitrites
Mix in a test tube 2.0 ml of the solution (of the sample or of a standard) with 1.0 ml of water and 3.0 ml of the mixture of colour reagents, shake and keep it in the dark at room temperature; after 30 min measure the absorbance at 540 nm against water.
-
Determination of the nitrates
Incubate a portion of the sample solution (and of the nitrate standards) with the imidazole buffer, the NADPH/FAD solution and the solution of nitrate reductase in accordance with clause 6 of the standard, and then carry out the colour reaction as for the nitrites.
-
Calibration curves and calculations
Plot the absorbances of the standards against the mass of nitrites (mg in 200 ml). Read off the content of nitrites in the sample and the total content after the reduction; calculate the nitrates from the difference, expressing the results in mg of NaNO2 and NaNO3 per kg.
-
Test report
Give the identification of the sample, the reference to EN 12014-3, the results for the nitrites and the nitrates, the date and the departures from the standard; the precision data from Annex A.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Homogeniser | A high-speed rotary knife or a mincer with a plate with holes ≤ 4.5 mm | — |
| Water bath | Maintaining 100 °C | — |
| pH meter | With a glass and a reference electrode, for setting the pH to 8.0–8.5 | — |
| Spectrometer | Measurement at 540 nm; glass or plastic cells without absorption at 540 nm | — |
| Fluted filter papers | Free of nitrates and nitrites | — |
| Volumetric flasks of 200 ml and 500 ml, pipettes, test tubes | For the standard solutions and the colour reaction | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Sulfanilamide | 63-74-1 | Colour reagent no. 1: 8 g in 500 ml of water on a bath, after cooling 330 ml of hydrochloric acid 37 % (free of nitrites), make up to 1 l; stable for several weeks |
| N-(1-naphthyl)ethylenediamine dihydrochloride | 1465-25-4 | Colour reagent no. 2: 0.330 g in 250 ml of water, in a dark bottle, replaced every week; the mixture of the reagents 1 + 1 (v/v) on the day of use |
| Carrez solutions | 14459-95-1 | No. 1: 150 g of K4[Fe(CN)6]·3H2O in 1 l (a dark bottle, replaced every two months); no. 2: 230 g of zinc acetate dihydrate (CAS 5970-45-6) in 1 l |
| Sodium nitrite | 7632-00-0 | A stock solution of 400 mg/l (200 mg to 0.1 mg in 500 ml; 2 weeks at 4 °C), diluted to 20 mg/l |
| Potassium nitrate | 7757-79-1 | A stock solution of 600 mg/l (300 mg in 500 ml), diluted to 30 mg/l |
| Imidazole, NADPH-Na4, FAD, nitrate reductase | 288-32-4 | The buffer: 0.68 g of imidazole in 80 ml of water, pH 7.8 with hydrochloric acid 2 mol/l, to 100 ml; the solution of NADPH with FAD prepared immediately before use; the enzyme (Aspergillus sp., EC 1.6.6.2) — available as the test kit „Nitrat"; sodium hydroxide of about 1 mol/l |
Health and safety (OHS)
- Hydrochloric acid 37 % in the colour reagent — a fume hood, goggles, gloves
- N-(1-naphthyl)ethylenediamine dihydrochloride and sodium nitrite — avoid raising dust and contact with the skin; sodium nitrite is toxic if swallowed
- The boiling water bath and the homogenisation with a hot extract — protection against scalding
- The Carrez reagents contain hexacyanoferrate — do not acidify the waste (a risk of the release of hydrogen cyanide)
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN 12014-3:2006 — “Foodstuffs — Determination of nitrate and/or nitrite content — Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite”.
How does this method work?
A homogenised sample (a temperature below 25 °C; a mincer with holes ≤ 4.5 mm, passed through twice) in an amount of 10 g (to 10 mg) is homogenised with about 50 ml of water for 30–60 s, rinsed with 50 ml of hot water, the pH is brought to 8.
What is the measuring range and accuracy?
Range of the validation: 9–22 mg/kg of nitrites (NaNO2) and 23–48 mg/kg of nitrates (NaNO3); applicability 5–125 mg/kg of the sum as NaNO2; Test portion and extraction: 10 g (to 10 mg), 50 ml of water + 50 ml of hot water, pH 8.0–8.5, 15 min in a boiling bath, a 200 ml flask; Clarification: 4 ml each of Carrez solutions no. 1 and no. 2; a fluted filter paper, the first 20 ml of the filtrate to be discarded; Colour reaction: 2.0 ml of the solution + 1.0 ml of water + 3.0 ml of the mixture of reagents, 30 min in the dark, 540 nm against water.
How long does the test take?
The times given for the individual stages add up to approximately 46 min–46 min. The procedure comprises 9 steps.
What equipment is required?
Homogeniser, Water bath, pH meter, Spectrometer, Fluted filter papers, Volumetric flasks of 200 ml and 500 ml, pipettes, test tubes.
Where is this test used?
Control of the content of nitrites and nitrates (E249–E252) in cured meats, canned goods and delicatessen products; Official supervision of the limits of additives in meat products; Studies of curing and of the transformations of nitrates during the ripening of cured meats; A testing procedure of the QAFP quality system for canned meat; A cadmium-free method — an alternative to cadmium reduction in laboratories limiting toxic waste.
What safety precautions apply?
Hydrochloric acid 37 % in the colour reagent — a fume hood, goggles, gloves; N-(1-naphthyl)ethylenediamine dihydrochloride and sodium nitrite — avoid raising dust and contact with the skin; sodium nitrite is toxic if swallowed; The boiling water bath and the homogenisation with a hot extract — protection against scalding; The Carrez reagents contain hexacyanoferrate — do not acidify the waste (a risk of the release of hydrogen cyanide).
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN 12014-3.