🍎 Determination of nitrate and nitrite in meat products by ion-exchange chromatography — 10 g of sample is homogenized with 50 ml of water at 50–60 °C, 50 ml of acetonitrile is added, made up to 200 ml and filtered through a fluted filter and a 0.45 µm membrane; the extract is separated on an anion exchanger with a lithium borate gluconate mobile phase with acetonitrile (pH 6.5) and measured at 205 nm; validation: nitrate 50–300 mg/kg, nitrite approx. 40 mg/kg, PN-EN 12014-4

Food chemistry PN-EN 12014-4

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN 12014-4:2006 (Polish version), in full (our translation): “An ion-exchange chromatographic (IC) method is described for the determination of nitrate and nitrite content in meat products, validated for various meat products with a nitrate content of 50 mg/kg to 300 mg/kg expressed as nitrate ion and a nitrite content of about 40 mg/kg, as nitrite ion”. We read the text of the standard in the sample of SIST EN 12014-4:2005 (the text of EN 12014-4:2005 without changes) from the title page through the contents, foreword and Clauses 1–5 up to and including the beginning of 6.5 (expression of results, formula 1). Outside the sample remain the explanations of formula (1), the formula for nitrate, Clause 7 (precision) and Annexes A–C (example chromatogram, precision data, further applicability).

ACCORDING TO THE TEXT OF EN 12014-4:2005 (Clauses 1–5, 6.1–6.5). The standard was prepared by CEN/TC 275 “Food analysis — Horizontal methods” (secretariat DIN); it superseded ENV 12014-4:1998. The EN 12014 series comprises: Part 1 — general considerations, Part 2 — HPLC/IC of nitrate in vegetables, Part 3 — spectrometry of nitrate and nitrite in meat products after enzymatic reduction (entry on PN-EN 12014-3), Part 4 — this method, Part 5 — enzymatic determination of nitrate in vegetable-containing baby food, Part 7 — continuous flow determination of nitrate in vegetables after cadmium reduction. Scope (1): ion-exchange chromatographic method for nitrate and nitrite in meat products, validated for various meat products at nitrate contents of 50–300 mg/kg (as nitrate ion) and nitrite of about 40 mg/kg (as nitrite ion); note: according to interlaboratory data the method may also be applied to nitrate in vegetables and baby food and to nitrite in meat products above 40 mg/kg. Normative reference (2): EN ISO 3696. Principle (3): nitrate and nitrite are extracted from the sample with hot water, the solution is treated with acetonitrile to remove interfering substances, and the content is determined by ion-exchange chromatography with UV detection at 205 nm. Reagents (4): reagents of recognized analytical grade and water of at least grade 3; acetonitrile; glycerol; lithium hydroxide anhydrous or monohydrate; boric acid 99 %; hydrochloric acid 1.8 mol/l (15 ml of acid 1.18 g/ml to 100 ml) and approx. 0.1 mol/l; stock solution — 1.500 g of sodium nitrite and 3.258 g of potassium nitrate in 1000 ml of water, usable for 2 weeks in a refrigerator at 4 °C (sodium nitrite is hygroscopic); standard solutions — 0; 0.1; 0.5; 1.0; 1.5 and 2.0 ml of stock solution to 200 ml, i.e. 0; 0.5; 2.5; 5.0; 7.5 and 10 mg/l of nitrite ion and 0; 1; 5; 10; 15 and 20 mg/l of nitrate ion, preferably prepared on the day of use; gluconic acid solution — 50 g to 100 ml (dark brown is clarified on SPE columns to slightly yellow); lithium borate gluconate buffer — 34.00 g of boric acid and 19.6 ml of gluconic acid solution in 500 ml of water, 11.00 g of anhydrous (or 19.26 g of monohydrate) lithium hydroxide, 125 ml of glycerol, to 1000 ml, stable 6 months at approx. 4 °C; mobile phase — 17 ml of buffer and 125 ml of acetonitrile in 1000 ml of water, pH 6.5 ± 0.1 (with hydrochloric acid 1.8, then 0.1 mol/l), filtered through a membrane of approx. 0.22 µm, stable not longer than one week, and the pH is checked every day before use — outside the range a new solution is prepared. Apparatus (5): homogenizer — high-speed rotational cutter or mincer with a plate with holes not exceeding 4.5 mm; magnetic stirrer (optional); fluted filters free of nitrate and nitrite — some filters absorb nitrate; membranes approx. 0.45 µm and 0.22 µm; SPE columns RP C18 (optional); isocratic liquid chromatograph with UV detector at 205 nm; anion exchange column 4.6 mm × 150 mm, polymethacrylate resin with quaternary ammonium group, particle size 10 µm, capacity (30 ± 3) µeq/ml, with a pre-column of approx. 20 mm. Preparation (6.1): homogenization such that the sample temperature does not exceed 25 °C; with a mincer at least twice; 10 g to the nearest 10 mg into a 150 ml conical flask. Extraction (6.2): the determination within one working day; 50 ml of water at 50–60 °C, thorough mixing with the homogenizer, washings into the flask, quantitative transfer into a 200 ml volumetric flask, 50 ml of acetonitrile, gentle mixing, cooling, making up with water; filtration through a fluted filter, then through a 0.45 µm membrane — a clear solution may be injected, a turbid one is filtered further through 0.22 µm; blank with 10 ml of water instead of the sample. Calibration (6.3): the standard solutions are injected, then the blank; linearity is checked. Conditions (6.4.1, for the column of 5.10.2): mobile phase of 4.12, UV 205 nm, injection volume at least 40 µl, flow 1 ml/min; with other columns the conditions are adjusted. Measurement (6.4.2): first standards, then blank and samples; in a series one standard every five samples; a peak outside the curve — dilute the solution with mobile phase and repeat; nitrate and nitrite are identified by the retention times of the standards, the content is read from the curve; the blank shows whether there was nitrate or nitrite contamination. Result (6.5): nitrite content in mg of ion per kg — formula (1): w(NO2−) = 200 × A(NO2−) / m × F (we did not read the explanation of the symbols).

STATUS (catalogue cards as of 03.10.2026). The card of PN-EN 12014-4:2006 (Polish version, published 24-07-2006, 18 pages, price group J) has no “Withdrawn” header: Food, Agriculture and Forestry Sector, KT 235 Food Analysis, ICS 67.120.10, “Introduces: EN 12014-4:2005 [IDT]”, “Supersedes: PN-EN 12014-4:2005 — English version”, “Additional elements: PN-EN 12014-4:2006/Ap1:2007” (we did not read the corrigendum Ap1). The card of PN-EN 12014-4:2005 (English version) — “Withdrawn” 24-07-2006, “Replaced by: PN-EN 12014-4:2006 — Polish version”; both introduce the same EN 12014-4:2005. In the iTeh catalogue (read 03.10.2026) the Slovenian adoption SIST EN 12014-4:2005 has the status “Published”.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 12014-4 appears in 6 records at 5 laboratories: AB 452, AB 545, AB 546, AB 583, AB 705. In the current scopes on the PCA website (read 03.10.2026, issues from 14.10.2025 to 06.05.2026) all give “PN-EN 12014-4:2006”, and AB 583 and AB 705 additionally “PN-EN 12014-4:2006/Ap1:2007”. The “Laboratories” tab (“PN-EN 12014-4”) shows the same 5 laboratories; checked with the tab query on the production server labcoda.pl on 03.10.2026.

WHAT THE LABORATORIES TEST (PCA items). Meat and meat products — all five; AB 452 also ready-to-eat dishes and fruit and vegetable products. Ranges and form of expression differ: AB 546 — nitrate (10–400) mg/kg, nitrite (5–200) mg/kg, ion-exchange chromatography with DAD detection; AB 583 — nitrate (20–300) mg/kg, nitrite (10–100) mg/kg, ion chromatography; AB 705 — sodium nitrite (2–120) mg/kg and sodium nitrate (2–250) mg/kg, ion chromatography; AB 545 — nitrite (5.0–500) mg/kg and nitrate (10.0–500) mg/kg, HPLC-UV; AB 452 — nitrite as NaNO2 (1.0–500) mg/kg and nitrate as NaNO3 (10–5000) mg/kg, HPLC-UV. The standard gives the result in mg of ion per kg (6.5) — ranges expressed as the sodium salt (AB 452, AB 705) are a different unit than the result of the standard. The lower limits for nitrite (from 1.0 to 10 mg/kg) lie below the level of about 40 mg/kg for which the method was validated, and the note to Clause 1 extends it to nitrite above 40 mg/kg, not below.

WHERE A SEEMINGLY CORRECT RESULT IS EASY. At preparation: the standard sets two conditions explicitly — the sample must not warm above 25 °C during homogenization (6.1), and the determination is completed within one working day (6.2); an extract left until the next day is a deviation from the method, and the result shows nothing of it (the standard gives no reason in the part read). At filtration: filters that absorb or contain nitrate lower or raise the result — hence filters “free of nitrate and nitrite” and the blank (5.4, 6.2, 6.4.2). At the mobile phase: the pH is checked daily, and a phase outside 6.5 ± 0.1 or older than one week is prepared anew (4.12) — peaks are identified only by the retention times of the standards (6.4.2), so a standard every five samples is the only check in the series. At measurement: interfering substances are removed by acetonitrile added to the extract (3, 6.2); a peak outside the calibration curve must be diluted with mobile phase and measured again, not extrapolated (6.4.2). At the result: formula (1) gives mg of NO2− ion per kg; the same sample expressed as NaNO2 gives a larger number by the ratio of molar masses — the report must state in which form the result is given.

WHAT WE DO NOT GIVE. We did not read the explanations of formula (1), the formula for nitrate, Clause 7 and the annexes, so we do not give the meaning of factor F, precision data or further applications of Annex C. We do not know the content of the corrigendum Ap1:2007. We do not give permitted amounts of nitrate and nitrite in meat products, because we did not read the texts of regulations for this entry.

Method principle

Nitrate and nitrite are washed out of the comminuted meat product with hot water, and acetonitrile added to the extract precipitates and removes interfering components. The filtrate is separated on an anion exchange column with a mobile phase of lithium borate gluconate buffer and acetonitrile (pH 6.5); both ions absorb UV radiation at 205 nm, and their content is read from a calibration curve, identifying the peaks by the retention times of the standards.

Applications

• Meat products — nitrate (validation 50–300 mg/kg of ion) and nitrite (approx. 40 mg/kg of ion, application above 40 mg/kg) (Clause 1)
• Nitrate in vegetables and baby food — according to interlaboratory data (note to Clause 1)
• Meat and meat products, ready-to-eat dishes, fruit and vegetable products — in the accreditation scopes of 5 laboratories at PCA

Key parameters

Standard

Standard number

PN-EN 12014-4:2006

Title (PL)

Artykuły żywnościowe — Oznaczanie zawartości azotanów i/lub azotynów — Część 4: Oznaczania zawartości azotanów i azotynów w produktach mięsnych metodą chromatografii jonowymiennej (IC)

Title (EN)

Foodstuffs — Determination of nitrate and/or nitrite content — Part 4: Ion-exchange chromatographic (IC) method for the determination of nitrate and nitrite content of meat products

Step-by-step procedure

1. Homogenization and test portion

   Sample not warmer than 25 °C, with the mincer at least twice; 10 g to the nearest 10 mg. PN-EN 12014-4:2006 current (PKN catalogue card as of 03.10.2026).

2. Extraction and clean-up

   50 ml of water 50–60 °C, homogenization, into a 200 ml flask, 50 ml of acetonitrile, cool, make up to the mark; fluted filter, 0.45 µm membrane (if needed 0.22 µm); blank with 10 ml of water. All within one day.

3. Calibration

   Six standard solutions (0–10 mg/l NO2−, 0–20 mg/l NO3−), then the blank; check linearity.

4. IC measurement

   UV 205 nm, ≥ 40 µl, 1 ml/min; a standard every five samples; identification by retention times; a peak outside the curve — dilute with mobile phase.

5. Result

   mg of ion per kg according to formula (1); explanation of the symbols and precision — outside the sample read.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Acetonitrile is flammable and toxic — work in a fume hood; sodium nitrite is toxic and oxidizing
• Lithium hydroxide and hydrochloric acid are corrosive — gloves and goggles; the standard in the part read contains no separate warnings

Frequently asked questions