🍄 Aflatoxins B1, B2, G1 and G2 in Nuts, Figs and Paprika — HPLC with Immunoaffinity Clean-up

Food Chemistry PN-EN 14123

Overview

STATUS (as of 02.11.2026). PN-EN 14123:2008 — Polish version, published on 12.09.2008, implements EN 14123:2007 [IDT]; replaced the English version PN-EN 14123:2008, which had replaced PN-EN 14123:2004; the PKN catalogue card has no withdrawal note (read on 02.11.2026). EN 14123:2007 replaced EN 14123:2003 — the only change was the inclusion of validation data for hazelnuts. The description is based on the preview of EN 14123:2007 (iTeh, SIST EN 14123:2008: clauses 1–5 and 6.1–6.3) and on the interlaboratory study on which the method is based (J. Stroka et al., J. AOAC Int. 2000, 83: 320–340).

SCOPE (clause 1). Determination of aflatoxins B1, B2, G1 and G2 in hazelnuts, figs, pistachios, peanuts and paprika powder. The limit of quantification is 0.8 ng/g for each aflatoxin or less (a value from intralaboratory and interlaboratory studies), depending on the instrumentation. The standard covers neither ochratoxin A nor aflatoxin M1; aflatoxin B1 in processed cereal-based foods for infants and young children is determined according to a separate standard, PN-EN 15851:2012 (implements EN 15851:2010).

PRINCIPLE (clause 3). The test portion is extracted with a mixture of methanol and water, or with this mixture with hexane (or cyclohexane) added. The extract is filtered, diluted with phosphate-buffered saline (PBS) and applied to an immunoaffinity column (IAC) containing antibodies specific to aflatoxins B1, B2, G1 and G2; the aflatoxins are eluted from the column with methanol. They are determined by RP-HPLC with post-column derivatization by bromination and fluorescence detection; the bromine is generated electrochemically or supplied as pyridinium hydrobromide perbromide (PBPB). The interlaboratory study of 2000 describes the same procedure: extraction with methanol and water (8 + 2) — for peanuts and pistachios with hexane or cyclohexane — dilution with PBS, washing of the column with water, elution with methanol and post-column bromination in an electrochemical cell (with bromide in the mobile phase) or with PBPB.

REAGENTS (clause 4). Water of grade 3 according to EN ISO 3696, for HPLC grade 1. PBS of pH 7.4: 0.20 g of potassium chloride, 0.20 g of potassium dihydrogen phosphate, 1.16 g of disodium hydrogen phosphate (or 2.92 g of the dodecahydrate) and 8.00 g of sodium chloride in 0.9 l of water, pH 7.4, made up to 1 l (tablets with equivalent properties are permitted). Extraction mixture: 8 parts by volume of methanol and 2 parts of water; n-hexane or cyclohexane; sodium chloride. IAC: capacity of at least 100 ng of aflatoxin B1 and a recovery of at least 80 % for B1, B2 and G1 and at least 60 % for G2 (aqueous standard solution with 10 % methanol, 5 ng of each toxin); the applied solution may contain at most 12 % methanol. Mobile phase A (with PBPB): water, acetonitrile and methanol 6 : 2 : 3; mobile phase B (with electrochemical bromine): the same mixture with 120 mg of potassium bromide and 350 µl of nitric acid 4 mol/l per litre. Post-column reagent: 50 mg of PBPB in 1 l of water (up to four days in the dark at room temperature). Aflatoxin stock solutions of 10 µg/ml each in toluene with acetonitrile (98 + 2); the concentration is determined from the absorption spectrum from 330 nm to 370 nm: ρ = Amax · M · 100 / (ε · b), where M and ε: B1 312 g/mol and 1930 m²/mol, B2 314 and 2040, G1 328 and 1660, G2 330 and 1790. The five calibration solutions contain from 0.400 ng/ml to 3.600 ng/ml of aflatoxin B1 and G1 and from 0.080 ng/ml to 0.720 ng/ml of B2 and G2 (Table 2).

APPARATUS (clause 5). Glassware in contact with aqueous aflatoxin solutions is acid-washed (e.g. with sulfuric acid 2 mol/l for several hours) — glassware not washed with acid causes losses of aflatoxins. A laboratory mill or an explosion-proof high-speed blender (for pastes and for the extraction of nuts and figs), a shaker (for paprika), fluted filter papers, a glass microfibre filter (1.6 µm or less), a 75 ml reservoir and a pump for the IAC, optionally a vacuum manifold. HPLC: pump for a flow rate of 1.0 ml/min; injection by complete filling of the loop, a 100 µl loop is recommended (with another loop the limit of detection at most 0.2 ng/g and the limit of quantification at most 0.5 ng/g for each aflatoxin); RP column, e.g. C18 or ODS-2, 25 cm × 4.6 mm, 5 µm, separating the aflatoxin peaks to baseline (overlap below 10 %); a PBPB derivatization system (pulse-free pump, zero-dead-volume T-piece, PTFE reaction tubing at least 45 cm × 0.5 mm) — only with phase A — or an electrochemical bromine generation system — only with phase B; a fluorescence detector with a 360 nm excitation filter and a 420 nm cut-off emission filter, and for detectors with a monochromator 365 nm (excitation), 435 nm (emission) and a bandwidth of 18 nm are recommended. In addition, 0.45 µm disposable filters (checked for losses of aflatoxins), an analytical balance (0.1 mg) and a laboratory balance (0.01 g).

PROCEDURE (clauses 6.1–6.3). A suitable quantity (e.g. 10 kg) of pistachios, peanuts, hazelnuts and figs is homogenized to a paste; the sample size and sampling are laid down in EU legislation. The IAC is brought to room temperature and conditioned with 10 ml of PBS passed at a rate of 2 ml/min to 3 ml/min, leaving about 0.5 ml above the packing. Pastes are extracted with a high-speed blender (fatty pastes must form an emulsion, and fig paste must disintegrate in the solvent); paprika ground to particles of at most 500 µm can be extracted by shaking. Hazelnuts: about 50 g, 4 g of sodium chloride and 100 ml of water — 1 min of blending; then 150 ml of methanol — 2 min; 5 ml of filtrate (equivalent to 1 g of sample) with 15 ml of PBS onto the column. Figs: about 50 g, 5 g of sodium chloride and 300 ml of extraction mixture — 3 min; 10 ml of filtrate with 60 ml of PBS. Peanuts and pistachios: about 50 g, 5 g of sodium chloride, 200 ml of extraction mixture and 100 ml of n-hexane or cyclohexane — 3 min; after filtration the lower phase, 10 ml with 60 ml of PBS (pistachios with strong turbidity: 20 ml of filtrate with 140 ml of PBS, filtration, 70 ml onto the column). Paprika: about 50 g, 5 g of sodium chloride and 300 ml of extraction mixture — vigorous shaking by hand for 15 s to 30 s, then 30 min on the shaker; 10 ml of filtrate with 60 ml of PBS. Test portions are weighed to the nearest 0.1 g; larger test portions are permitted if the ratio of sample to solvent is kept.

REGULATIONS. Maximum levels of aflatoxins are laid down in Regulation (EU) 2023/915 (Annex I, point 1.1), which repealed Regulation (EC) No 1881/2006; for the matrices of this standard (aflatoxin B1 / sum of B1, B2, G1 and G2, µg/kg): dried figs 6.0 / 10.0; groundnuts (peanuts) to be subjected to sorting 8.0 / 15.0, for the final consumer 2.0 / 4.0; pistachios (with almonds and apricot kernels) to be subjected to sorting 12.0 / 15.0, for the final consumer 8.0 / 10.0; hazelnuts (with Brazil nuts) to be subjected to sorting 8.0 / 15.0, for the final consumer 5.0 / 10.0; paprika and other spices of the genus Capsicum 5.0 / 10.0. For the sum of aflatoxins the levels refer to lower-bound concentrations — values below the limit of quantification are taken as zero.

WHAT WE DO NOT GIVE HERE. The clean-up on the IAC (washing and elution volumes), the chromatography and derivatization conditions, the calculations and the precision data (6.4–7, Annex B) — these parts of the standard are not in the preview. In the interlaboratory study of 2000 (16 laboratories; samples spiked with 2.4 ng/g and 9.6 ng/g of total aflatoxins) the recovery of total aflatoxins was from 71 % to 92 %, and that of aflatoxin B1 from 82 % to 109 %.

Method principle

A test portion of the sample (about 50 g) is extracted with a mixture of methanol and water (8 + 2) with sodium chloride — peanuts and pistachios with n-hexane or cyclohexane added, hazelnuts first with water, then with methanol. The filtrate is diluted with PBS buffer (at most 12 % methanol) and passed through an immunoaffinity column on which antibodies bind aflatoxins B1, B2, G1 and G2; the aflatoxins are eluted with methanol.

The eluate is separated by reversed-phase HPLC. After the column the aflatoxins are brominated — with bromine generated electrochemically (potassium bromide and nitric acid in the mobile phase) or with a solution of pyridinium hydrobromide perbromide — and detected with a fluorescence detector (excitation 360–365 nm, emission 420–435 nm). The result is given for aflatoxin B1 and for the sum of the four aflatoxins.

Applications

• Hazelnuts, peanuts, pistachios, figs and paprika powder — aflatoxin B1 and the sum of aflatoxins B1, B2, G1 and G2 (clause 1)
• Checking the maximum levels of aflatoxins from Regulation (EU) 2023/915 (Annex I, points 1.1.3–1.1.11, 1.1.14) in these products
• Processed cereal-based foods for infants and young children (aflatoxin B1) — separate standard PN-EN 15851:2012; ochratoxin A and aflatoxin M1 — outside the scope of this standard

Key parameters

Standard

Standard number

PN-EN 14123:2008

Title (PL)

Artykuły żywnościowe — Oznaczanie aflatoksyny B1 oraz sumy aflatoksyn B1, B2, G1 i G2 w orzechach laskowych, orzechach ziemnych, pistacjach, figach i papryce w proszku — Metoda wysokosprawnej chromatografii cieczowej z uzyskiwaniem pochodnej po rozdziale na kolumnie i oczyszczaniu na kolumnie powinowactwa immunologicznego

Title (EN)

Foodstuffs — Determination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1 and G2 in hazelnuts, peanuts, pistachios, figs, and paprika powder — High performance liquid chromatographic method with post-column derivatisation and immunoaffinity column cleanup

Step-by-step procedure

1. Homogenization of the sample

   A suitable quantity (e.g. 10 kg) of nuts and figs is made into a paste with a high-speed blender; paprika is ground to particles of at most 500 µm (6.1, 6.3.1).

2. Conditioning of the IAC

   Column at room temperature; 10 ml of PBS at a rate of 2 ml/min to 3 ml/min, about 0.5 ml of PBS remains above the packing (6.2).

3. Extraction

   About 50 g with sodium chloride: hazelnuts — 100 ml of water (1 min) and 150 ml of methanol (2 min); figs — 300 ml of methanol with water 8 + 2 (3 min); peanuts and pistachios — 200 ml of the mixture and 100 ml of hexane or cyclohexane (3 min); paprika — 300 ml of the mixture, 30 min on the shaker (6.3.2–6.3.6).

4. Dilution of the filtrate

   Hazelnuts: 5 ml of filtrate with 15 ml of PBS; others: 10 ml of filtrate with 60 ml of PBS; pistachios with precipitation: 20 ml of filtrate with 140 ml of PBS, filtration, 70 ml onto the column (6.3).

5. Clean-up, HPLC and result

   Diluted extract onto the IAC, washing with water, elution with methanol; RP-HPLC with post-column bromination and fluorescence detection; calibration with five solutions (Table 2); details in 6.4–7 of the standard, outside the preview (clause 3; 2000 study).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Laboratory waste containing aflatoxins is decontaminated according to the procedures developed by the International Agency for Research on Cancer (IARC) (warning 1 in 4.19)
• Aflatoxins are decomposed by light: the laboratory is protected from daylight (UV-absorbing foil, subdued light), and solutions are stored in the dark, in aluminium foil or amber glass, at the temperature recommended by the manufacturer, e.g. −18 °C (warning 2 in 4.19)
• Methanol, acetonitrile, hexane and cyclohexane are flammable, and methanol is toxic — follow the safety data sheets; the standard does not address all hazards, safety measures are established by the user (warning in the foreword)

Frequently asked questions