🍎 Nitrate in vegetables and vegetable products — HPLC/IC

Food Chemistry PN-EN 12014-2

Overview

STATUS (as of 03.11.2026). PN-EN 12014-2:2018-01 — Polish version (published on 26.11.2020; earlier an English version of 2018), implements EN 12014-2:2017 [IDT] and replaced PN-EN 12014-2:2001, withdrawn on 17.01.2018. In the iTeh catalogue, EN 12014-2:2017 has the status “Published” and stage 90.93 — decision to confirm the standard after systematic review. The standard does not cover meat or meat products: nitrate and nitrite in meat products are described in separate Knowledge Base entries according to PN-EN 12014-3 (enzymatic reduction and spectrometry) and PN-EN 12014-4 (ion-exchange chromatography).

CHANGES FROM THE 1997 EDITION (foreword). The lower limit of application was reduced from 50 to 25 mg/kg nitrate and the upper limit was deleted, purification methods 1 and 2 used in the preparation of the test solutions were deleted, iceberg lettuce was added to the interlaboratory test, the HPLC and IC conditions and chromatograms were updated, and the procedure was extensively revalidated.

SCOPE AND PRINCIPLE (clauses 1, 3 and introduction). The standard specifies an HPLC method and an IC method for the determination of nitrate in vegetables and vegetable products with a content of 25 mg/kg or greater. It was validated on naturally contaminated and spiked samples: beetroot juice (194 and 691 mg/kg), puréed carrots (26 and 222 mg/kg) and iceberg lettuce (623 and 3542 mg/kg). The method was developed for investigations regarding EU legislation on nitrate in vegetables; according to the introduction, it is also suitable for the determination of nitrite in other matrices, which was not validated in the interlaboratory test. Nitrate is extracted with hot water and determined by reversed-phase HPLC with UV detection or by ion chromatography with conductivity or UV detection.

SAMPLE PREPARATION AND EXTRACTION (6.1–6.3). Liquid samples (e.g. vegetable juices) are shaken vigorously and, if required, filtered through a fluted filter paper or prediluted with water to about 25 mg/l nitrate. From solid samples (e.g. leaf vegetables), adhering animals, soil and dirty particles and, if required, inedible or damaged external leaves are removed; the sample is shredded (e.g. in a laboratory cutter) and homogenised; the homogenate is analysed immediately or frozen at once and thawed as gently as possible (e.g. overnight in the refrigerator). If a fresh sample cannot be homogenised within 24 h, the whole sample is frozen and analysed within a maximum of 6 weeks; the requirements of EU legislation are taken into account — a footnote of the standard gives an example of provisions under which samples are not washed. Pasty samples (e.g. mashed vegetables) are stirred repeatedly until homogeneous. About 10 g of a solid or pasty sample (to the nearest 1 mg) are covered in a 500 ml conical flask with about 400 ml of hot water (about 80 °C) and, after the bath has boiled again, kept for 15 min in a boiling water bath, cooled, transferred to a 500 ml volumetric flask, made up to the mark, shaken and filtered through a fluted filter paper. The solutions are filtered through a membrane filter if necessary (at least 15 ml, discarding the first few millilitres) and then dialysed or injected directly; at least one reagent blank is run in parallel.

CALIBRATION AND CALCULATION (6.4, clause 7). The calibration curve is usually prepared from five concentration levels and a blank, from standard solutions preferably of 1–50 mg/l prepared from a 1000 mg/l NO₃⁻ stock solution; equal volumes of sample and standard solutions are injected, and a solution whose response is outside the curve is diluted with water and analysed again. Result: w (mg/kg) or ρ (mg/l) = x × F × 1000 × V / m, where x is the concentration from the curve minus the reagent blank (mg/l), F the factor of any further dilution, V the volume of the extract (0.5 l) and m the sample mass or volume; the result is reported without decimal places, according to current legislation.

PRECISION (7.3). Repeatability limits r and reproducibility limits R from the interlaboratory test, e.g. beetroot juice 194 mg/kg — r 14 mg/kg, R 51 mg/kg; puréed carrots 26 mg/kg — r 3.3 mg/kg, R 12 mg/kg; iceberg lettuce 3542 mg/kg — r 105 mg/kg, R 876 mg/kg.

REGULATIONS. Commission Regulation (EU) 2023/915 (Annex I section 6.1, consolidated text of 19.08.2026) sets maximum levels for nitrate in mg NO₃/kg: fresh spinach 3500, preserved, deep-frozen or frozen spinach 2000, fresh lettuce 3000–5000 (depending on growing under cover or in the open air and on the harvest period), iceberg lettuce 2500 (grown under cover) or 2000 (open air), rucola 7000 (harvested from 1 October to 31 March) or 6000 (from 1 April to 30 September), baby foods and processed cereal-based foods for infants and young children 200.

WHAT WE DO NOT GIVE HERE. The example HPLC and IC conditions (Annexes C and D: columns, mobile phases, injection volumes), the chromatograms (Annex A) and the full precision data (Annex B) — they are outside the available text of the standard.

Method principle

Nitrate is extracted from the sample with hot water in a boiling water bath. The extract is filtered and nitrate is determined by reversed-phase HPLC with UV detection or by ion chromatography with conductivity or UV detection. The content is calculated from the calibration curve after subtracting the reagent blank and reported in mg/kg or mg/l without decimal places.

Applications

• Nitrate in vegetables and vegetable products at contents from 25 mg/kg (clause 1)
• Vegetable juices, leaf vegetables and vegetable purées (6.1)
• Control of maximum levels for nitrate in spinach, lettuce, iceberg lettuce and rucola (Regulation (EU) 2023/915, Annex I section 6.1)

Key parameters

Standard

Standard number

PN-EN 12014-2:2018-01

Title (PL)

Artykuły żywnościowe — Oznaczanie zawartości azotanów(V) i/lub azotanów(III) — Część 2: Oznaczanie zawartości azotanów(V) w warzywach i przetworach warzywnych metodą HPLC/IC

Title (EN)

Foodstuffs — Determination of nitrate and/or nitrite content — Part 2: HPLC/IC method for the determination of nitrate content of vegetables and vegetable products

Step-by-step procedure

1. Liquid samples

   Shake a representative sample vigorously; if required, filter it through a fluted filter paper or predilute it with water to about 25 mg/l nitrate (6.1.1).

2. Solid and pasty samples

   Remove dirt and inedible leaves, shred and homogenise; analyse immediately or freeze and thaw gently. Stir pasty samples until homogeneous (6.1.2, 6.1.3).

3. Hot water extraction

   Weigh about 10 g (to the nearest 1 mg) into a 500 ml conical flask, add about 400 ml of water at about 80 °C, keep for 15 min in a boiling water bath after it has boiled again, cool, transfer to a 500 ml volumetric flask, make up, shake and filter through a fluted filter paper (6.2).

4. Test solution

   If necessary, filter through a membrane filter (at least 15 ml, discard the first millilitres), dialyse or inject directly; run at least one reagent blank (6.3).

5. Calibration and determination

   Prepare a curve from usually five standard solutions and a blank; inject equal volumes of sample and standard solutions, identify nitrate by its retention time and dilute a solution outside the curve with water (6.4, 6.5, 7.1).

6. Calculation

   w or ρ = x × F × 1000 × V / m (V = 0.5 l); report the result in mg/kg or mg/l without decimal places (7.2).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Sulfuric acid 96 % — in the classification notified to ECHA: causes severe skin burns and eye damage (H314)
• The available text of the standard contains no safety clause — other health and safety rules are set by the laboratory

Frequently asked questions